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Localization and characterization of phosphodiesterase II in intestinal mucosa Flanagan, Peter Rutledge 1974

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LOCALIZATION AND CHAPACTERIZATION OF PHOSPHODIESTERASE IN INTESTINAL MUCOSA by PETER R. FLANAGAN B . S c , U n i v e r s i t y C o l l e g e D u b l i n , 1967 M . S c , U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1970 A THESIS SUBMITTED I N PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n t h e D e p a r t m e n t o f B i o c h e m i s t r y 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 t h e r e q u i r e d s t a n d a r d _ THE UNIVERSITY OF B R I T I S H A u g u s t , 1974 COLUMBIA In presenting th i s thesis in par t i a l fu l f i lment of the requirements for an advanced degree at the Univers i ty of B r i t i s h Columbia, I agree that the L ibrary shal l make it f ree ly ava i lab le for reference and study. I further agree that permission for extensive copying of th is thesis for scholar ly purposes may be granted by the Head of my Department or by his representat ives. It is understood that copying or pub l i cat ion of th is thesis for f inanc ia l gain shal l not be allowed without my written permission. Department The Univers i ty of B r i t i s h Columbia Vancouver 8, Canada i ABSTRACT PDase I I a c t i v i t y was d e t e r m i n e d u s i n g a s y n t h e t i c s u b s t r a t e , t h e 2 , 4 - d i n i t r o p h e n y l e s t e r o f t h y m i d i n e 3 ' - p h o s p h a t e . The enzyme a c t i v i t y was e s t i m a t e d i n f r a c t i o n s o b t a i n e d b y d i f f e r e n t i a l c e n t r i f u g a t i o n o f h o m o g e n a t e s o f e p i t h e l i a l c e l l s f r o m t . t h e s m a l l i n t e s t i n a l m u c o s a o f g u i n e a p i g s and r a t s . I n g u i n e a p i g p r e p a r a t i o n s P Dase I I o c c u r r e d w i t h h i g h e s t s p e c i f i c a c t i v i t y i n t h o s e f r a c t i o n s r i c h i n s u c c i n a t e d e h y d r o g e n a s e and a c i d p h o s p h a t a s e . A l y s o s o m a l l o c a t i o n f o r t h e g u i n e a p i g enzyme was i n d i c a t e d b y i t s s t r u c t u r e - l i n k e d l a t e n c y and b y i t s a s s o c i a t i o n w i t h p a r t i c l e s w h i c h u n d e r w e n t a c h a r a c t e r -i s t i c d e c r e a s e i n e q u i l i b r i u m d e n s i t y when T r i t o n WR-1339 was i n j e c t e d i n t o t h e a n i m a l s . W i t h r a t p r e p a r a t i o n s a much g r e a t e r p r o p o r t i o n o f t h e PDase I I a c t i v i t y was f o u n d i n t h e s o l u b l e f r a c t i o n a f t e r u u l t - r a ; c ; e n t r i f u g a t i o n . The r a t enzyme e x h i b i t e d a l o w e r d e g r e e o f l a t e n c y a nd a d m i n -i s t r a t i o n o f T r i t o n WR-1339 h a d no e f f e c t . The r a t enzyme a c t i v i t y i n t h e s e c r u d e p r e p a r a t i o n s f u r t h e r d i f f e r e d f r o m t h a t o f t h e g u i n e a p i g i n o t h e r r e s p e c t s ; i t was more l a b i l e a t 60°C, e x h i b i t e d a s l i g h t l y l o w e r pH o p t i m u m , h a d a h i g h e r m o l e c u l a r w e i g h t as d e t e r m i n e d b y g e l f i l t r a t i o n c h r o m a t o g r a p h y and d i s p l a y e d a much s m a l l e r t e n d e n c y t o a g g r e g a t e u n d e r L l o w s a l t c o n d i t i o n s . B o t h enzymes were p u r i f i e d b y c h r o m a t o g r a p h y on D E A E - c e l l u l o s e , C M - c e l l u l o s e a nd a g a r o s e , t h e e x t e n s i v e p u r i f i c a t i o n (550 f o l d ) o f t h e r a t enzyme b e i n g l a r g e l y due t o i t s b e h a v i o u r o h t h e l a t t e r m a t e r i a l w h e r e i t was f o u n d t o b i n d t e n a c i o u s l y i n l o w i o n i c s t r e n g t h s o l u t i o n s . On t h e o t h e r h a n d , o n l y a f i f t e e n - f o l d p u r i f i c a t i o n o f t h e g u i n e a p i g enzyme was o b t a i n e d b e c a u s e o f i t s t e n d e n c y t o f f o r m i n s o l u b l e a g g r e g a t e s d d u r i n g t h e c h r o m a t o g r a p h i c s t e p s . I n t h e m a i n , t h e p r o p e r t i e s o f t h e p a r t i a l l y p u r i f i e d enzymes w e r e q u i t e s i m i l a r . B o t h d i s p l a y e d pH o p t i m a b e t w e e n pH 6 and 7, w e r e i n h i b i t e d i n s o l u t i o n s o f h i g h i o n i c s t r e n g t h , w e r e u n a f f e c t e d ' b y d i v a l e n t c a t i o n s o r EDTA, w e r e s i m i l a r l y i n a c t i v a t e d b y h e a t i n g a t a temp-e r a t u r e o f 60°G d i s p l a y e d d i s c o n t i n u o u s A r r h e n i u s p l o t s _5 and e x h i b i t e d K m v a l u e s o f t h e o r d e r 2-5x10 M f o r dTpDNP. I n m o s t c a s e s t f c h e d i f f e r e n c e s b e t w e e n t h e enzymes w e r e j u s t d i f f e r e n c e s o f d e g r e e and c o u l d p r o b a b l y be a c c o u n t e d f o r b y e t h e d i f f e r e n t e x t e n t s t o w h i c h t h e enzymes w e r e p u r i f i e d . A more e x t e n s i v e c h a r a c t e r i z a t i o n o f t h e h i g h l y p u r i f i e d r a t PDase was c a r r i e d o u t . The f a l l - o f f i n PDase I I r e a c t i o n r a t e o b s e r v e d a t h i g h enzyme l e v e l s w i t h dTpDNP a s s u b s t r a t e was f o u n d t o be due t o c o m p e t i t i v e i n h i b i t i o n o f t h e enzyme by dTp, a r e a c t i o n p r o d u c t w h i c h showed a o f 2x10 M. The i s o e l e c t r i c p o i n t o f PDase I I was e s t i m a t e d by e l e c t r o f o c u s i n g b u t s i n c e m u l t i p l e p e a k s o f a c t i v i t y w e r e f o u n d a t pH 3.4, 4.2-4.5, a n d pH 7.2 a c o n c l u s i v e r e s u l t was n o t o b t a i n e d . P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s o f p u r i f i e d r a t PDase I I i n d i c a t e d t h a t t h e p a t t e r n o b t a i n e d was, i n p a r t , d e p e n d e n t on w h e t h e r t h e p r e p a r a t i o n was f r e s h o r n o t ; f r e s h l y p u r i f i e d PDase I I c o n t a i n e d up t o 10 b a n d s i n g e l s s t a i n e d f o r p r o t e i n w h e r e a s o n l y 1-2 b a n d s w e r e o b t a i n e d when t h e p r e p a r a t i o n s w e r e " a g e d " . A m o l e c u l a r w e i g h t o f 150000-170000 f o r t h e enzyme was e s t i m a t e d i n e x p e r i m e n t s p e r f o r m e d b y g e l - f i l t r a t i o n c h r o m a t o g r a p h y on d e x t r a n a n d a g a r o s e g e l s . I n v e s t i g a t i o n o f t h e i n t e r a c t i o n w i t h , a n d h y d r o l y s i s b y , r a t PDase I I o f a number o f p o s s i b l e p h o s p h o d i e s t e r s u b s t r a t e s i n d i c a t e d that'-, t h e enzyme r e q u i r e d a n u c l e o s i d e 3 ' - p h o s p h o r y l r e s i d u e f o r t h e i n i t i a t i o n o f h y d r o l y s i s w h i c h t h e n p r o c e e d e d i n a 5'+3' d i r e c t i o n . F i n a l l y , t h e e f f e c t o f some enzyme i n h i b i t o r s was i n v e s t i g a t e d . P D a se I I a c t i v i t y was i n h i b i t e d i n t h e p r e s e n c e ; o f NEM, PCMB, PCMPS a n d i o d o a c e t i c a c i d . I t was f u r t h e r f o u n d t h a t t h e i n a c t i v a t i o n b y i o d o a c e t i c a c i d c o u l d be p r e v e n t e d b y t h e p r e s e n c e o f a PDase s u b s t r a t e o r , b e t t e r s t i l l , b y dTp. T h i s i s g o o d e v i d e n c e t h a t i o d o a c e t a t e a l k y l a t e s an e s s e n t i a l r e s i d u e a t t h e a c t i v e c e n t e r o f PDase I I and i s t h e f i r s t t i m e t h a t s u c h an e f f e c t h a s b e e n shown f o r a PD a s e . i v TABLE OF CONTENTS Page ABSTRACT i TABLE OF CONTENTS .... . . i v L I S T OF TABLES i x L I S T OF FIGURES ....... .... . x i ABBREVIATIONS x i v ACKNOWLEDGEMENTS ................... x v INTRODUCTION . . . . . . •'. 1 D e f i n i t i o n 1 C l a s s i f i c a t i o n a n d n o m e n c l a t u r e o f n u c l e a s e s 1 D i v e r s i t y a n d f u n c t i o n o f n u c l e a s e s i n v i v o 6 R e a s o n s f o r s t u d y i n g n u c l e a s e s 10 P h o s p h o d i e s t e r a s e I I 11 M e a s u r e m e n t o f p h o s p h o d i e s t e r a s e I I a c t i v i t y . 14 I n t e s t i n a l p h o s p h o d i e s t e r a s e s 16 The p r e s e n t i n v e s t i g a t i o n 17 MATERIALS AND METHODS 21 MATERIALS 21 C h e m i c a l s 21 A n i m a l s 22 METHODS-METHODS . . . . . . . . . . . . - . . . . .... 23 DEAEC c h r o m o t o g r a p h y 23 CMC c h r o m a t o g r a p h y 25 A g a r o s e c h r o m a t o g r a p h y 26 V Page D N A - c e l T u l o s e c h r o m a t o g r a p h y . . §7 G e l f i l t r a t i o n c h r o m a t o g r a p h y 2 8 F l o w r a t e s i n c o l u m n c h r o m a t o g r a p h y 29 U l t r a f i l t r a t i o n .. 30 P a p e r c h r o m a t o g r a p h y o f n u c l e o t i d e s 30 I d e n t i f i c a t i o n o f 3 1 - n u c l e o t i d e s ... 31 P r e p a r a t i o n and. c h a r a c t e r i z a t i o n o f t h y m i d i n e 3 1 - ( 2 , 4 - d i n i t r o p h e n y l ) p h o s p h a t e ; 31 P r e p a r a t i o n o f e p i t h e l i a l c e l l s u s p e n s i o n s 37 S u b c e l l u l a r f r a c t i o n a t i o n 38 S o n i c a t i o n o f p a r t i c u l a t e f r a c t i o n s 41 D e n s i t y - g r a d i e n t c e n t r i f u g a t i o n o f p a r t i c u l a t e f r a c t i o n s 41 D e n s i t y - g r a d i e n t c e n t r i f u g a t i o n o f " s o l u b i l i z e d " g u i n e a p i g a n d r a t PDase I I 42 Ammonium s u l p h a t e f r a c t i o n a t i o n .... 42 Zone p r e c i p i t a t i o n 43 E x t r a c t i o n o f r a t i n t e s t i n a l PDase I I 44 E x t r a c t i o n o f g u i n e a p i g i n t e s t i n a l P Dase I I 45 " A g i n g " o f p u r i f i e d r a t PDase I I ... 46 E l e c t r o f o c u s i n g 47 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 . 49 D e t e c t i o n o f p r o t e i n i n p o l y a c r y l a -m i d e g e l s :. . 50 v i P a g e D e t e c t i o n o f c a r b o h y d r a t e i n p o l y a c r y l a m i d e g e l s 50 D e t e c t i o n o f PDase I I a c t i v i t y i n p o l y a c r y l a m i d e g e l s 50 L i g h t m i c r o s c o p y 51 E l e c t r o n m i c r o s c o p y 52 Enzyme d e t e r m i n a t i o n s 53 Enzyme u n i t s 5 6 A n a l y t i c a l m e t h o d s 56 RESULTS AND DISCUSSION 5 8 PART A. THE SUBCELLULAR LOCATION OF INTESTINAL PDASE I I 58 D i f f e r e n t i a l c e n t r i f u g a t i o n o f e p i t h e l i a l c e l l h o m o g e n a t e s 58 A c t i v a t i o n o f a c i d p h o s p h a t a s e and PDase I I 64 E f f e c t o f T r i t o n WR-13 39 o n t h e e q u i l i b r i u m d e n s i t y o f p a r t i c u l a t e PDase I I 64 Some p r o p e r t i e s o f g u i n e a p i g a n d r a t PDase I I 71 I n f l u e n c e o f pH 74 H e a t s t a b i l i t y 76 M o l e c u l a r s i z e 81 D i s c u s s i o n 83 PART B. P U R I F I C A T I O N OF INTESTINAL PDASE I I 87 E x p e r i m e n t s u s i n g r a t p r e p a r a t i o n s 87 P r e c i p i t a t i o n b y ammonium s u l p h a t e . 89 Zone p r e c i p i t a t i o n 92 v i i P a ge A d s o r p t i o n o n a g a r o s e 9 5 I o n e x c h a n g e c h r o m a t o g r a p h y 98 P u r i f i c a t i o n o f t h e r a t enzyme 99 D N A - c e l l u l o s e c h r o m a t o g r a p h y 103 E x p e r i m e n t s u s i n g g u i n e a p i g p r e p a r a t i o n s .... 103 E x t r a c t i o n o f g u i n e a p i g i n t e s t i n a l P Dase I I . . . 103 P u r i f i c a t i o n o f t h e g u i n e a p i g enzyme 108 Some p r o p e r t i e s o f p a r t i a l l y p u r i f i e d p h o s p h o d i e s t e r a s e I I f r o m t h e g u i n e a p i g a n d t h e r a t 113 pH o p t i m u m 113 The e f f e c t o f v a r i o u s compounds on PDase I I a c t i v i t y 114 H e a t s t a b i l i t y 119 E f f e c t o f dTpDNP c o n c e n t r a t i o n 122 E f f e c t o f t e m p e r a t u r e o n t h e h y d r o l y -s i s o f dTpDNP 124 D i s c u s s i o n 128 P u r i f i c a t i o n o f t h e enzyme 12 8 C o m p a r i s o n o f p a r t i a l l y p u r i f i e d r a t and g u i n e a p i g i n t e s t i n a l P D a s e I I . 131 PART C. A STUDY OF THE PROPERTIES OF RAT INTESTINAL PDASE I I 134 The h y d r o l y s i s o f dTpDNP 135 pH r e q u i r e m e n t s 137 M o l e c u l a r w e i g h t 137 I s o e l e c t r i c p o i n t 142 v i i i P age G e l e l e c t r o p h o r e s i s 143 C o n t a m i n a t i n g enzyme a c t i v i t i e s . . .'. 148 H y d r o l y s i s o f p h o s p h o d i e s t e r s 151 E f f e c t o f p h o s p h o d i e s t e r s o n dTpDNP h y d r o l y s i s 155 E f f e c t o f p h o s p h o m o n e s t e r s 157 E f f e c t o f d i v a l e n t c a t i o n s 159 E f f e c t o f enzyme i n h i b i t o r s 159 P r o t e c t i o n a g a i n s t t h e e f f e c t o f i o d a c e t a t e 169 C o m p a r i s o n o f two PDase I I a c t i v i t i e s o b t a i n e d b y D N A - c e l l u l o s e chromofeography 169 D i s c u s s i o n 172 i x L I S T OF TABLES T a b l e Page I P o s s i b l e c r i t e r i a f o r n u c l e a s e c l a s s i f i c a t i o n .... 3 I I S i t e s o f a c t i o n o f a number o f DNA r e s t r i c t i o n enzymes 8 I I I Some s u b s t r a t e s f o r PDase I I 12-13 I V H y d r o l y s i s o f dTpDNP i n v a r i o u s b u f f e r s 36 V C o m p o s i t i o n o f t h e s o l u t i o n s u s e d i n t h e e l e c t r o f o c u s i n g e x p e r i m e n t s 4 8 V I S u b c e l l u l a r d i s t r i b u t i o n o f c o m p o n e n t s i n homo-g e n a t e s o f e p i t h e l i a l c e l l s f r o m g u i n e a p i g and r a t i n t e s t i n e s -59 V I I D i s t r i b u t i o n o f PDase I I , a c i d p h o s p h a t a s e a n d p r o t e i n i n h o m o g e n a t e s o f g u i n e a p i g i n t e s t i n a l e p i t h e l i a l c e l l s p r e p a r e d i n m e d i a o f d i f f e r e n t v i s c o s i t y 63 V I I I The d e g r e e o f a c t i v a t i o n o f PDase I I a n d a c i d p h o s p h a t e b y T r i t o n X-100 i n p a r t i c u l a t e f r a c t i o n s o f i n t e s t i n a l e p i t h e l i a l c e l l s 65 I X T e s t f o r a d s o r p t i o n o f s o l u b l e PDase I I b y p a r t i c u l a t e f r a c t i o n s 88 X Ammonium s u l p h a t e p r e c i p i t a t i o n o f r a t i n t e s t i n a l P Dase I I 91 X I P u r i f i c a t i o n o f r a t i n t e s t i n a l PDase I I 102 X I I D N A - c e l l u l o s e c h r o m a t o g r a p h y o f p u r i f i e d r a t PDase I I 105 X I I I C o m p a r i s o n o f t h e b e h a v i o u r o f f r e s h a n d " a g e d " p r e p a r a t i o n s o f p u r i f i e d r a t PDase I I o n D N A - c e l l u l o s e 105 X I V S o l u b i l i z a t i o n o f g u i n e a p i g PDase I I 107 X W P u r i f i c a t i o n o f PDase f r o m g u i n e a p i g a n d r a t i n t e s t i n e 112 X T a b l e Page XVI The e f f e c t o f s u c c i n a t e c o n c e n t r a t i o n on t h e a c t i v i t y o f p u r i f i e d r a t and g u i n e a p i g PDase I I .. 116 X V I I The e f f e c t s o f u r e a a nd v a r i o u s s a l t s o n r a t and g u i n e a p i g PDase a c t i v i t y 117 X V I I I C o m p a r i s o n o f t h e i n a c t i v a t i o n o f c r u d e a n d p u r i f i e d r a t PDase T I a t 60°C 120 X I X PDase I I a c t i v i t y i n a v a r i e t y o f b u f f e r s 138 XX E f f e c t o f p r o n a s e a n d t r y p s i n on PDase I I a c t i v i t y 149 X X I C o n t a m i n a t i n g enzyme a c t i v i t i e s i n p u r i f i e d p r e p a r a t i o n s o f r a t i n t e s t i n a l P Dase I I 150 X X I I The r a t e o f h y d r o l y s i s o f some compounds c o n t a i n i n g p h o s p h o d i e s t e r b o n d s by p u r i f i e d PDase I I 153 X X I I I I E f f e c t s o f p h o s p h o d i e s t e r s o n t h e r a t e o f dTpDNP h y d r o l y s i s b y p u r i f i e d PDase I I 156 X X I V E f f e c t o f some p h o s p h o m o n e s t e r s on t h e r a t e o f dTpDNP h y d r o l y s i s by p u r i f i e d PDase 158 XXV E f f e c t o f d i v a l e n t c a t i o n s o n PDase I I a c t i v i t y ... 162 XXVI E f f e c t o f a number o f p o s s i b l e enzyme i n h i b i t o r s .. 164 X X V I I P o l y n u c l e o t i d a s e a c t i v i t y o n PDase I I f r a c t i o n s p u r i f i e d o n D N A - c e l l u l o s e c h r o m a t o g r a p h y 171 x i L I S T OF FIGURES F i g u r e Page 1. A d i a g r a m m a t i c r e p r e s e n t a t i o n o f t h e s m a l l i n t e s t i n e i n l o n g i t u d i n a l s e c t i o n 19 2. S e p a r a t i o n o f dTpDNP f r o m w a t e r - s o l u b l e r e a c t a n t s by DEAEC c h r o m a t o g r a p h y 33 3. The s p e c t r a o f dTpDNP and i t s b a s e - c a t a l y s e d h y d r o l y s i s p r o d u c t s 35 4. The i s o l a t i o n o f i n t e s t i n a l e p i t h e l i a l c e l l s ...... 39 5. L o c a l i z a t i o n o f PDase and o t h e r c o m p o n e n t s i n s u b c e l l u l a r f r a c t i o n s o b t a i n e d b y d i f f e r e n t i a l c e n t r i f u g a t i o n o f i n t e s t i n a l e p i t h e l i a l c e l l h o m o g e n a t e s 61 6. S u c r o s e d e n s i t y - g r a d i e n t c e n t r i f u g a t i o n o f " m i t o c h o n d r i a l - l y s o s o m a l " f r a c t i o n s f r o m g u i n e a p i g l i v e r h o m o g e n a t e s 67 7. S u c r o s e d e n s i t y - g r a d i e n t c e n t r i f u g a t i o n o f f r a c t i o n s I I I + I V f r o m g u i n e a p i g i n t e s t i n a l e p i t h e l i a l c e l l h o m o g e n a t e s 6 8 8. F r a c t i o n a t i o n o f f r a c t i o n s I I I + I V f r o m g u i n e a p i g i n t e s t i n a l e p i t h e l i a l c e l l h o m o g e n a t e s on a s h a l l o w s u c r o s e d e n s i t y - g r a d i e n t 70 9. E l e c t r o n m i c r o s c o p y o f s u b c e l l u l a r p a r t i c l e s f r o m t h e i n t e s t i n a l e p i t h e l i a l c e l l s o f a n u n i n j e c t e d g u i n e a p i g 72 10. E l e c t r o n m i c r o s c o p y o f s u b c e l l u l a r p a r t i c l e s f r o m t h e i n t e s t i n a l e p i t h e l i a l c e l l s o f a g u i n e a p i g w h i c h h a d b e e n i n j e c t e d w i t h T r i t o n WR-1339 73 11 . I n f l u e n c e o f pH o n t h e a c t i v i t y o f r a t a n d g u i n e a p i g PDase I I 75 12. The s t a b i l i t y o f r a t i n t e s t i n a l PDase I I a c t i v i t y a t v a r i o u s pH v a l u e s 77 13. The s t a b i l i t y o f r a t and g u i n e a p i g PDase I I a c t i v i t y a t 60°C 78 14. I n f l u e n c e o f pH o n t h e a c t i v i t y o f h e a t - t r e a t e d g u i n e a p i g PDase I I 79 x i i F i g u r e Page , 15. V a r i a t i o n i n t h e s t a b i l i t y o f g u i n e a p i g PDase I I a c t i v i t y a t 60°C . 80 16. S u c r o s e d e n s i t y - g r a d i e n t c e n t r i f u g a t i o n o f f r a c t i o n V I f r o m r a t a n d g u i n e a p i g e p i t h e l i a l c e l l h o m o g e n a t e s 82 17. M o l e c u l a r w e i g h t d e t e r m i n a t i o n o f i n t e s t i n a l P D ase I I f r o m t h e r a t a n d g u i n e a p i g b y g e l f i l t r a t i o n 84 18. /Ammonium s u l p h a t e f r a c t i o n a t i o n o f t h e s u p e r n a t a n t f r a c t i o n o b t a i n e d f r o m a r a t i n t e s t i n a l h o mogenate 90 19. The p r i n c i p l e o f z o n e p r e c i p i t a t i o n 93 20. Zone p r e c i p i t a t i o n o f r a t i n t e s t i n a l PDase I I ..... 94 21. C h r o m a t o g r a p h y o f r a t i n t e s t i n a l PDase I I o n a g a r o s e . . . .T 9 6 22. C h r o m a t o g r a p h i c p u r i f i c a t i o n o f r a t i n t e s t i n a l P Dase I I 100 23. D N A - c e l l u l o s e c h r o m a t o g r a p h y o f p u r i f i e d r a t PDase I I 104 24. DEAEC c h r o m a t o g r a p h y o f r a t a n d g u i n e a p i g . i n t e s t i n a l e x t r a c t s 109 25 . CMC c h r o m a t o g r a p h y o f p a r t i a l l y p u r i f i e d r a t and g u i n e a p i g PDase I I 110 26. C h r o m a t o g r a p h y o f p a r t i a l l y p u r i f i e d r a t a n d g u i n e a p i g PDase I I on a g a r o s e I l l 27. I n f l u e n c e o f pH on t h e a c t i v i t y o f p u r i f i e d r a t and g u i n e a p i g PDase I I 115 28. H e a t i n a c t i v a t i o n o f c r u d e a n d p u r i f i e d r a t and g u i n e a p i g PDase I I 121 29. E f f e c t o f s u b s t r a t e c o n c e n t r a t i o n on p u r i f i e d r a t and g u i n e a p i g PDase I I 123 30. E f f e c t o f s u b s t r a t e c o n c e n t r a t i o n on p a r t i a l l y p u r i f i e d r a t and g u i n e a p i g PDase I I 12 5 x i i i F i g u r e Page 31. A r r h e n i u s p l o t s o f t h e r a t and g u i n e a p i g PDase I I c a t a l y s e d h y d r o l y s i s r e a c t i o n s 127 32. The r e l e a s e o f DNP f r o m dTpDNP by p u r i f i e d PDase I I 136 33. The a c t i v i t y o f r a t PDase I I as a f u n c t i o n o f t h e amount o f enzyme p r e s e n t 138 34. E f f e c t o f M g C l 2 , E D T A a n d ( N H 4 ) 2 S 0 4 o n t h e pH o p t i m u m o f r a t PDase I I 140 35. M o l e c u l a r w e i g h t e s t i m a t i o n o f r a t i n t e s t i n a l P Dase I I b y g e l f i l t r a t i o n c h r o m a t o g r a p h y 141 36. E l e c t r o f o c u s i n g o f p u r i f i e d r a t i n t e s t i n a l P Dase I I 144 37. P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s o f f r e s h l y p u r i f i e d r a t PDase I I 145 38. 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 o s i s o f a p u r i f i e d p r e p a r a t i o n o f r a t PDase I I w h i c h h a d b e e n " a g e d " . 147 39. The h y d r o l y s i s o f GpA by p u r i f i e d r a t PDase I I .... 152 40. C o m p e t i t i v e i n h i b i t i o n o f p u r i f i e d r a t PDase I I by GpA a n d Tp 160 41. D i x o n p l o t o f t h e ' i n h i b i t i o n o f PDase I I b y Tp .... 161 42. C u m u l a t i v e e f f e c t o f PCMPS and m e r c a p t o e t h e n o l o n r a t PDase I I a c t i v i t y 166 43. I n a c t i v a t i o n o f PDase I I b y i o d o a c e t i c a c i d 167 44. The pH d e p e n d e n c e o f i o d o a c e t a t e i n a c t i v a t i o n o f PDase I I 168 45. P r o t e c t i o n o f PDase I I a g a i n s t i n a c t i v a t i o n b y i o d o a c e t i c a c i d 170 x i v ABBREVIATIONS A a d e n o s i n e A c i d p h o s . a c i d p h o s p h a t a s e AMP a d e n o s i n e m o n o p h o s p h a t e ATP a d e n o s i n e 5 1 - t r i p h o s p h a t e C c y t i d i n e CM c a r b o x y m e t h y l CMC c a r b o x y m e t h y l c e l T u l o s e DCC d i c y c l o h e x y l c a r - b o d i i m i d e D ' P SEE: d i e t h y T a m i ' n o e f e h y l DEAEC d i e t h y l a m i n o e t h y l c e l l u l o s e dN d e o x y n u c l e o s i d e DNA d e o x y r i b o n u c l e i c a c i d DNAase d e o x y r i b o n u c l e a s e DNP 2 , 4 - d i n i t r o p h e n o l dTMP d e o x y t h y m i d i n e m o n o p h o s p h a t e dTpDNP d e o x y t h y m i d i n e 3 1 - ( 2 f 4 - d i n i t r o p h e n y l ) p h o s p h a t e dTpNP d e o x y t h y m i d i n e 3 1 - ( p - n i t r o p h e n y l ) p h o s p h a t e EDTA e t h y l e n e d i a m i n e t e t r a a c e t i c a c i d g g r a v i t a t i o n a l f o r c e c a l c u l a t e d u s i n g t h e maximum r a d i u s o f r e v o l u t i o n o f t h e c e n t r i f u g e r o t o r . G guano s i n e G-6-Pase g l u c o s e - 6 - p h o s p h a t a s e K a v d i s t r i b u t i o n c o e f f i c i e n t i n g e l f i l t r a t i o n c h r o m a t o g r a p h y Kj_ i n h i b i t o r c o n s t a n t K m M i c h a e l i s c o n s t a n t MES 2 - ( N - m o r p h o l i n o ) e t h a n e s u l p h o n i c a c i d mRNA m e s s e n g e r r i b o n u c l e i c a c i d N n u c l e o s i d e NEM N - e t h y l m a l e i m i d e Np n u c l e o s i d e 3 ' - p h o s p h a t e NP p - n i t r o p h e n o l NpN a d i n u c l e o s i d e m o n o p h o s p h a t e ( c a n be e i t h e r 2'-^5' o r 3'~*75') PAS p e r i o d i c a c i d s c h i f f s PCA p e r c h l o r i c a c i d PCMB p - c h l o r o m e r c u r i b e n z o a t e PCMPS p - c h l o r o m e r c u r i p h e n y l s u l p h o n i c a c i d PDase p h o s p h o d i e s t e r a s e P i o r t h o p h o s p h a t e pN n u c l e o s i d e 5 ' - p h o s p h a t e p o l y A p o l y a d e n y l i c a c i d RNA r i b o n u c l e i c a c i d RNAase r i b o n u c l e a s e SDH s u c c i n a t e d e h y d r o g e n a s e T t h y m i d i n e TEMED N,N,N * ' , N ' t e t r a m e t h y l - e t h y l e n e - d i a m i n e T r i s 2 - a m i n o - 2 - h y d r o x y m e t h y l p r o p a n e - l , 3 - d i o l u v u l t r a v i o l e t V m a Y maximum i n i t i a l v e l o c i t y XV ACKNOWLEDGEMENTS I w o u l d l i k e t o e x p r e s s my s i n c e r e s t t h a n k s t o my r e s e a r c h s u p e r v i s o r , D r . S. H. Z b a r s k y f o r h i s k i n d n e s s , p a t i e n c e , e n c o u r a g e m e n t , g u i d a n c e a n d c r i t i c i s m d u r i n g my s e v e n y e a r s o j o u r n i n h i s l a b o r a t o r y . I a l s o w i s h t o t h a n k t h e o t h e r members o f my Ph.D. c o m m i t t e e , D r s . C. T. B e e r a n d M. S m i t h f o r t h e i r d i r e c t i o n a n d i n t e r e s t . S p e c i a l t h a n k s a r e a l s o due t o D r s . P. D. B r a g g , W. J . P o l g l a s e , G. M. T e n e r , M. D a r r a c h a n d M. S m i t h f o r t h e u s e o f e q u i p m e n t , some o f w h i c h , a t t i m e s , m u s t h a v e a p p e a r e d t o them t o be on p e r m a n e n t " l o a n " ; t o D r s . R. P o u l s o n a n d I . C. G i l l a m f o r t h e i r h e l p a n d a d v i c e i n many d i s c u s s i o n s ; t o Ms. E. Lo f o r p e r f o r m i n g t h e e l e c t r o n m i c r o s c o p y and t o Mr. V. W y l i e f o r t h e p r e p a r a t i o n o f s l i d e s f o r l i g h t m i c r o s c o p y . My w a r m e s t g r a t i t u d e i s a l s o due t o my p a r e n t s , e s p e c -i a l l y my m o t h e r , f o r r e a s s u r a n c e a n d s u p p o r t p a r t i c u l a r l y when s p i r i t s w e r e l o w . F i n a l l y , I w o u l d l i k e t o e x t e n d my h e a r t f e l t t h a n k s t o my w i f e f o r h e r l o v i n g u n d e r s t a n d i n g , p a t i e n c e a n d e n c o u r a g e m e n t e s p e c i a l l y d u r i n g t h e w r i t i n g o f t h i s t h e s i s . W i t h o u t h e r , t o paro d y t h e c l i c h e , n o n e o f t h i s w o u l d h a v e b e e n n e c e s s a r y ! The M e d i c a l R e s e a r c h C o u n c i l o f C a n a d a i s t h a n k e d f o r s t u d e n t s h i p s g r a n t e d f o r t h e p e r i o d 1968-1972 a nd t h e s u p p o r t o f t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a i n t h e fba?m o f G r a d u a t e F e l -l o w s h i p s f o r 1972-1974 i s a l s o a c k n o w l e d g e d . x v i K r e b s & V e e c h (1969) t e l l t h e p a r a b l e o f t h e b i o c h e m i s t who s e t o u t t o d i s c o v e r how a m o t o r c a r w o r k s . He h o m o g e n i s e d i t i n an a q u e o u s medium and i n a W a r b u r g manometer o b s e r v e d a r a p i d e v o l u t i o n o f g a s . H o p e f u l l y , he t h o u g h t t h a t t h i s g a s p r o d u c t i o n d r i v e s t h e c a r b u t e v e n t u a l l y r e a l i s e d t h a t i t was c a u s e d by t h e a c t i o n o f t h e b a t t e r y a c i d on t h e m e t a l o f t h e c a r b o d y . E x t e n d i n g t h e p a r a b l e , t h i s b i o c h e m i s t , who s p e c i a l i s e d i n i n t e r m e d i a r y m e t a b o l s i m , e n l i s t e d t h e a i d o f a c o l l e a g u e , a r e a l e n z y m o l o g i s t , who f r a c t i o n a t e d t h e c a r and i s o l a t e d t h e i n t a c t b a t t e r y . I n s u i t a b l e k i n e t i c e x p e r i m e n t s he showed t h a t i t w o u l d c a u s e a s m a l l w h e e l t o r e v o l v e , c o n c l u d e d t h a t t h e c a r was e l e c t r i c a l l y d r i v e n , and p r e d i c t e d t h a t a c a r e f u l s e a r c h b y h i s m e t a b o l i c c o l l e a g u e w o u l d r e v e a l a v a r i a b l e r e s i s t a n c e b e n e a t h t h e a c c e l e r a t o r p e d a l . — D a l z i e l (1973) i INTRODUCTION D e f i n i t i o n A c c o r d i n g t o t h e R e c o m m e n d a t i o n s o f t h e Com-m i s s i o n on B i o c h e m i c a l N o m e n c l a t u r e on t h e N o m e n c l a t u r e and C l a s s i f i c a t i o n o f Enzymes (1972) , PDase I I i s d e f i n e d a s c a t a l y s i n g " t h e e x o l y t i c h y d r o l y s i s o f 5 ' - h y d r o x y l -t e r m i n a t e d o l i g o n u c l e o t i d e s t o 3 ' - m o n o n u c l e o t i d e s " . I t s s y s t e m a t i c name i s o l i g o n u c l e a t e 3 1 - n u c l e o t i d o h y d r o l a s e and t h e c l a s s i f i c a t i o n number a s c r i b e d t o t h e enzyme i s 3.1.4.18. Thus by d e f i n i t i o n t h e n a t u r a l s u b s t r a t e s f o r P Dase I I a r e p o l y n u c l e o t i d e s and t h e enzyme i s t h e r e f o r e a p o l y n u c l e o t i d a s e ( R a z z e l l , 1 9 6 7 ) . C l a s s i f i c a t i o n a nd n o m e n c l a t u r e o f n u c l e a s e s A l t h o u g h t h e r e a r e o n l y 2 m a i n c l a s s e s o f n u c l e i c a c i d s (DNA and RNA), a p l e t h o r a o f enzymes i s known w h i c h c a n h y d r o l y s e t h e s e . B e c a u s e cr- t h e s p e c i f i c i t y o f t h e s e n u c l e a s e s , a s t h e y w e r e o r i g i n a l l y c a l l e d b y I w a n o f f ( 1 9 0 3 ) , v a r i e s a g r e a t d e a l , some d i f f i c u l t y h a s b e e n e n c o u n t e r e d i n d e v i s i n g a c o n s i s t e n t b u t s i m p l e . . j c l a s s i f i c a t i o n .• o f t h e s e e nzymes. The a r b i t r a r y a s s i g n m e n t o f names a n d / o r t h e u s e o f w o r d s a n d t e r m s w h i c h mean d i f f e r e n t t h i n g s t o d i f f e r e n t p e o p l e h a s e n s u r e d t h a t a t h o r o u g h l y c o n f u s i n g s i t u a t i o n now 2 e x i s t s i n t h i s f i e l d . S c h m i d t & L a s k o w s k i (1961) s e l e c t e d some . p r o p e r t i e s t h a t c o u l d be u s e d f o r t h e p u r p o s e o f c l a s s i f i c a t i o n . T h e s e c r i t e r i a , shown i n T a b l e I , a r e s t i l l u s e f u l d e s p i t e t h e g r e a t i n c r e a s e i n k n o w l e d g e a b o u t c h a r a c t e r i z a t i o n andr;»meonanism o f a c t i o n o f n u c l e a s e s w h i c h h a s o c c u r r e d s i n c e t h a t t i m e . T hus n u c l e o l y t i c enzymes c a n be c l a s s i f i e d a s RNAases o r DNAases and a s e x o - o r e n d o n u c l e a s e s . L a s k o w s k i a l s o i n t r o d u c e d t h e c o n c e p t o f u s i n g Roman n u m e r a l s i n o r d e r t o g r o u p enzymes w h i c h h a v e s i m i l a r p r o p e r t i e s ( Cunningham & L a s k o w s k i , 1 9 5 3 ) . Thus t h e two m o s t s t u d i e d DNAases o f t h a t t i m e , p a n c r e a t i c DNAase ( K u n i t z , 1950) and thymus DNAase (Maver & G r e c o , 1949) w e r e c a l l e d b y C u n n i n g h a m and L a s k o w s k i (1953) DNAase I and DNAase I I r e s p e c t i v e l y . The t e r m " i n t e s t i n a l DNAase I " t h e r e f o r e , i s u n d e r s t o o d t o mean "an enzyme w i t h an o p t i m a l pH, and i o n i c r e q u i r e m e n t s s i m i l a r t o t h a t o f p a n c r e a t i c DNAase I " ( L a s k o w s k i , 1 9 6 1 ) . The u s e o f t h i s scheme was e x t e n d e d b y R a z z e l l (1967) t o i n c l u d e enzymes w i t h an e x o n u c l e o l y t i c mode o f a c t i o n . The p r o t o t y p e s u s e d w e r e s n a k e venom PDase and s p l e e n PDase w h i c h he c a l l e d P D a s e s I and I I r e s p e c t i v e l y . U n f o r t u n a t e l y , s i n c e R a z z e l l (1961) a l s o u s e d t h e s e t e r m s t o d i s t i n g u i s h enzymes a c t i v e t o w a r d d i f f e r e n t s y n t h e t i c s u b s t r a t e s an l e x t r a c r i t e r i o n n o t i n t e n d e d b y C u n n i n g h a m and L a s k o w s k i (1953) was i n c o r p o r a t e d i n t o t h e c l a s s i f i c a t i o n ( R a z z e l l , 1 9 6 7 ) . 3 T a b l e I.. P o s s i b l e c r i t e r i a f o r n u c l e a s e c l a s s i f i c a t i o n . The s u g g e s t i o n s a r e t h o s e o f S c h m i d t & L a s k o w s k i Q. 9-61I . 1. T y p e o f s u b s t r a t e (DNA o r RNAL 2. Mode o f a c t i o n (Endo- o r e x o n u c l e a s e l 3. P r o d u c t s f o r m e d C5'-monoesterified o r 3 1 - m o n o e s t e r i f i e d I 4 U n d o u b t e d l y , f r o m t h i s s i t u a t i o n a r o s e t h e e r r o n e o u s b e l i e f t h a t 5 '-monophosphate a n d 3 1 - m o n o p h o s p h a t e f o r m e r s , ( s e e T a b l e I ) , a r e i d e n t i c a l w i t h t h e c l a s s I and I I enzymes r e f e r r e d t o a b o v e . To add t o t h e c o n f u s i o n , Roman n u m e r a l s h a v e a l s o b e e n u s e d i n an e n t i r e l y d i f f e r e n t d e s i g n a t i o n t o d e s c r i b e t h e DNAases o f E. c o l i (Lehman, 1 9 6 3 ) . I n t h i s s y s t e m t h e n u m e r a l s I , I I , I I I , e t c . r e f e r t o t h e t e m p o r a l o r d e r i n w h i c h t h e enzyme a c t i v i t i e s w e r e d i s c o v e r e d . T h u s e x o n u c l e a s e I I I was t h e t h i r d e x o n u c l e a s e a c t i v i t y i d e n t i f i e d i n E. c o l i . A f u r t h e r a n n o y i n g a m b i g u i t y l i e s i n t h e v a r i o u s m e a n i r i g s s a s c r i b e d t o t h e w o r d " p h o s p h o d i e s t e r a s e " . Uzawa (1932) o r i g i n a l l y u s e d t h e t e r m t o d i s t i n g u i s h enzymes c a p a b l e o f h y d r o l y s i n g d i e s t e r i f i e d p h o s p h a t e f r o m t h o s e t h a t s p e c i f i c a l l y a t t a c k e d m o n o p h o s p h a t e s (phosphomono-e s t e r a s e s ) . I n t i m e , h o w e v e r , t h e m e a n i n g o f t h e t e r m s h i f t e d t o become a s p e c i f i c name f o r enzymes t h a t a t t a c k e d o l i g o n u c l e o t i d e s and p r o d u c e d o n l y m o n o n u c l e o t i d e s . Thus t h e names venom p h o s p h o d i e s t e r a s e a n d s p l e e n p h o s p h o -d i e s t e r a s e d e n o t e s p e c i f i c r a t h e r t h a n g e n e r i c e n t i t i e s . I t was i n t h i s s e n s e t h a t R a z z e l l (1967) c o i n e d t h e t e r m s PDase I a n d PDase I I . Lehman (1960) p r o p o s e d t h a t " p h o s p h o d i e s t e r a s e s " be u s e d t o d e f i n e t h o s e enzymes w h i c h c a n o n l y a t t a c k s i n g l e - s t r a n d e d p o l y d e o x y n u c l e o t i d e c h a i n s a s o p p o s e d t o 5 DNAases w h i c h a r e s p e c i f i c f o r d o u b l e - s t r a n d e d DNA. However s u g g e s t i o n s h a v e b e e n made by S c h m i d t & L a s k o w s k i (1961) and e n d o r s e d by B e r n a r d i & B e r n a r d i (1968) t h a t t h e t e r m " p h o s p h o d i e s t e r a s e s " s h o u l d a g a i n be u s e d s e n s u s t r i c t u t o d e n o t e a c l a s s o f enzymes t h a t h y d r o l y s e p h o s p h o d i e s t e r b o n d s . Y e t a n o t h e r s o u r c e o f c o n f u s i o n a r i s e s f r o m t h e u s e , i n r e c e n t y e a r s , o f t h e t e r m s 3 ' - e x o n u c l e a s e and 5'-e x o n u c l e a s e . U s u a l l y t h o s e t e r m s a r e u n d e r s t o o d t o mean t h a t t h e a p p r o p r i a t e e x o n u c l e a s e i n i t i a t e s h y d r o l y s i s a t t h e 3'- o r 5'- end o f p o l y n u c l e o t i d e c h a i n s . H owever, i n some c a s e s r t h e t e r m s a r e a p p a r e n t l y u s e d t o d e s i g n a t e t h e t y p e o f p r o d u c t f o r m e d , e i t h e r 3'- o r 5 1 - m o n o n u c l e o t i d e s ( s e e Enzyme N o m e n c l a t u r e , p. 2 0 7 , E l s e v i e r S c i e n t i f i c P u b l i s h i n g Company, A m s t e r d a m , 197 3 ) . The n o m e n c l a t u r e o f t h e RNAases i s no l e s s d i s o r g -a n i s e d . B o t h Roman n u m e r a l s y s t e m s o u t l i n e d a b o v e h a v e b e e n u s e d w i t h t h e r e s u l t t h a t " a c i d RNAase I I " ( R a z z e l l , 1967) i s e q u i v a l e n t t o "RNAase I " (Rahman, 1 9 6 7 ) . I n a d d i t i o n many e n z y m e s , e s p e c i a l l y t h o s e f r o m f u n g a l s o u r c e s , a r e d e s i g n a t e d b y c a p i t a l l e t t e r s a s 'RNAase T^, RNAase T 2 e t c . ( U c h i d a & Egami., 1971) . I n v i e w o f t h e a b o v e d i s c u s s i o n , i t i s n o t s u r p r i s i n g t h a t a l e g i o n o f names h a s b e e n u s e d t o d e s c r i b e t h e enzyme t h a t a t t a c k s o l i g o n u c l e o t i d e c h a i n s f r o m t h e 5 1 - e n d y i e l d i n g 3 ' - n u c l e o t i d e s a s p r o d u c t s . Some o f t h e s e i n c l u d e s p l e e n 6 PDase (Hilmo'e, 1960) ; i n t e s t i n a l PDase ( H e p p e l & H i l m o e , 1 9 5 5 ) ; s p l e e n e x o n u c l e a s e ( B e r n a r d i & B e r n a r d i , 1 9 6 8 ) ; a c i d e x o n u c l e a s e (van Dyck & W a t t i a u x , 1 9 6 8 ) ; s p l e e n a c i d e x o n u c l e a s e ( B e r n a r d i & B e r n a r d i , 1 9 7 1 ) ; e x o n u c l e a s e ( p h o s p h o d i e s t e r a s e ) (Menon & S m i t h , 197 0 ) ; PDase I I ( R a z z e l l , 1 9 6 7 ) . The t e r m PDase I I w i l l be u s e d t h r o u g h o u t t h i s t h e s i s s i n c e i t i s t h e name recommended by t h e i n t e r n a t i o n a l a u t h o r i t y on s u c h m a t t e r s (see D e f i n i t i o n ) . D i v e r s i t y and f u n c t i o n o f n u c l e a s e s i n v i v o L e v e n e a t t r i b u t e d t h e f i r s t o b s e r v a t i o n o f n u c l e a s e a c t i v i t y t o w o r k e r s i n t h e l a b o r a t o r i e s o f K o s s e l / S a l k o w s k i and S c h u t z e n b e r g e r a l m o s t a c e n t u r y ago (Levene & M e d i g r e c e a n u , 1 9 1 1 a ) . However t h e f o u n d a t i o n o f p r e s e n t day i d e a s a c o n c e r n i n g t h e h y d r o l y s i s o f n u c l e i c a c i d s by d i f f e r e n t enzymes was l a i d by L e v e n e and M e d i g r e c e a n u (1911 a , b f c ) t h e m s e l v e s when t h e y t r e a t e d y e a s t and thymus n u c l e i c a c i d s w i t h i n t e s t i n a l j u i c e . They o b s e r v e d (a) a r a p i d f a l l i n t h e o p t i c a l r o t a t i o n o f t h e n u c l e i c a c i d s o l u t i o n and lb) a r a p i d l o s s o f t h e p r e c i p i t a b i l i t y o f n u c l e i c a c i d by s t r o n g m i n e r a l a c i d . L e v e n e b e l i e v e d t h a t t h r e e k i n d s o f enzymes were i n v o l v e d i n t h e breakdown o f n u c l e i c a c i d s , n amely n u c l e i n a s e , n u c l e o t i d a s e and n u c l e o s i d -a s e ( L e v e n e & M e d i g r e c e a n u , 1911c; L e v e n e & B a s s , 1931) b u t s i n c e t h o s e e a r l y d a y s a h o s t o f n u c l e a s e s has been s t u d i e d and c h a r a c t e r i z e d f r o m a v a r i e t y o f s o u r c e s . Most 7 i n t e r e s t i n g l y , the s p e c i f i c i t y of these ubiquitous enzymes appears to vary a great deal. For example, the r e s t r i c t i o n endonucleases of some b a c t e r i a l strains hydro-lyse unmodified DNA at a small number of unique s i t e s ; t h i s highly s p e c i f i c action r e s u l t s from the fact that the enzyme apparently cleaves the DNA at a point about'-'which there exists a two-fold "palindrome-like" axis of symmetry (Table I I ) . This .situation may be contrasted with the r e l a t i v e l y unspecific endo- and exonucleases which are present i n a l l organisms? With regard to the function of nucleases, i t i s probably safe to say that t h e i r diverse s p e c i f i c i t y , referred to above, precludes a common ro l e for these enzymes i n vivo. To say that RNAases and DNAases e x i s t to break down RNA and DNA respectively i s true but begs the question. In fact, the b i o l o g i c a l functioncof only a very •few of t h i s large group of enzymes i s presently known. Thus the role of r e s t r i c t i o n endonucleases i s rather obviously to protect the host bacterium from infectious DNA (Meselson et aJL. , 1972). Also the 5 '+31 and 3'-*-5* exonuclease a c t i v i t i e s associated with E. c o l i DNA polymerase are thought to p a r t i c i p a t e i n the synthetic repair of DNA by t h i s enzyme (Kornberg, 1969). In addition DNAases have been implicated i n DNA synthesis by the observation that sharp increases i n DNAase a c t i v i t y occur in c e l l s infected by some animal viruses (Lehman, 1967). Because the t o t a l 8 T a b l e I I . S i t e s o f a c t i o n o f a number o f DNA r e s t r i c t i o n e n z y m e s . The s e q u e n c e s w e r e t a k e n f r o m B a r r e l l & C l a r k . C1974). The a r r o w s i n d i c a t e t h e p o i n t s o f c l e a v a g e B y t h e e n z y m e s . Enzyme S e q u e n c e H. p a r a i n f l u e n z a e I 4-5'-GpTpTpApApC-3 1 • • • • • • 3'-CpApApTpTpG-5 1 H. p a r a i n f l u e n z a e I I 5'-NpCpCpGpGpN-3' 3'-NpGpGpCpCpN-5' E. c o l i R I 5 4- i 1-NpGpApApTpTpCpN-3 1 3 1 -N'pCpTpTpApApGpN-5 ' + E. c o l i R I I 5 4- » 1-NpCpCpApGpGpN-3 * 3 ' -N'pGpGpTpCpCpN-5 ' H. i n f l u e n z a e I I I 5'-ApApGpCpTpT-3' 3 1-TpTpCpGpApA-5' V 9 c e l l u l a r DNA r e m a i n e d c o n s t a n t f o r s e v e r a l h o u r s a f t e r i n f e c t i o n , a p u r e l y d e g r a d a t i v e r o l e f o r t h e s e i n d u c e d DNAases was c o n s i d e r e d u n l i k e l y . I n t h i s c o n n e c t i o n , A l l f r e y and M i r s k y (1952) compared t h e " a c i d " DNAase c o n t e n t o f v a r i o u s t i s s u e s o f c a l f , h o r s e , c h i c k e n , mouse and r a t and o b s e r v e d a c o r r e l a t i o n between t h e n u c l e a s e c o n t e n t o f a p a r t i c u l a r t i s s u e and i t s c a p a c i t y f o r p r o l i f e r a t i o n o r r e g e n e r a t i o n . B a r n a r d (1969a) has s u g g e s t e d a number o f f u n c t i o n s f o r RNAases: (a) r e m o v a l o f i n f o r m a t i o n a l RNA m o l e c u l e s ; (b) d e f e n s e a g a i n s t v i r a l RNA; (c) a g e n e r a l i n t r a c e l l u l a r d i g e s t i v e f u n c t i o n ; and (d) s y n t h e s i s o f p o l y n u c l e o t i d e s . A p r i m e c a n d i d a t e f o r t h e d e s t r u c t i o n o f mRNA i n E . c o l i h as b e en RNAase I I ( n u m e r o l o g y o f Lehman, 1963) as d i s c u s s e d by M i z u n o & A n r a k u ( 1 9 6 7 ) . A l s o RNAases o b v i o u s l y p l a y an i m p o r t a n t s p e c i f i c r o l e i n t h e p r o c e s s i n g o f p r e c u r s o r RNAs t o y i e l d s m a l l e r b i o l o g i c a l l y a c t i v e components (Burdon, 1971) . A l m o s t n o t h i n g i s known a b o u t t h e s p e c i f i c f u n c t i o n s o f n u c l e a s e s i n mammals o r i n h i g h e r o r g a n i s m s g e n e r a l l y . I t i s l i k e l y t h a t some o f t h e s e enzymes p e r f o r m an i n t r a -c e l l u l a r d i g e s t i v e f u n c t i o n e s p e c i a l l y s i n c e many o f t h o s e o f t h e a c i d t y p e have been shown by de Duve and c o w o r k e r s t o r e s i d e i n t h e l y s o s o m e s (de Duve, 1 9 6 3 ) . The h i g h l e v e l s o f some n u c l e a s e s i n p a r t i c u l a r t i s s u e s d o e s n o t a l w a y s i n d i c a t e an o b v i o u s f u n c t i o n , however. F o r i n s t a n c e , 10 a l t h o u g h b i o c h e m i s t r y t e x t b o o k s h a v e f o r many y e a r s i n d i c a t e d t h a t t h e f u n c t i o n - o f p a n c r e a t i c RNAase was t o h y d r o l y s e d i e t a r y RNA i n t h e i n t e s t i n e s o f man and o t h e r mammals, t h i s ' i s n o t c o m p l e t e l y c o r r e c t . The f u n c t i o n - ~ o f t h e h i g h c o n c e n t r a t i o n s o f enzyme f o u n d i n cows and o t h e r r u m i n a t i n g u n g u l a t e s h a s b e e n shown b y B a r n a r d (1969b) t o be r e l a t e d t o s p e c i a l i z e d f e a t u r e s o f r u m i n a n t m e t a b o l i s m . He o b s e r v e d : (a) t h e enzyme l e v e l was h i g h e s t i n r u m i n a n t s w h e r e t h e d i e t a r y RNA c o n t e n t was l o w ; (b) t h e enzyme l e v e l was v e r y l o w i n man w h e r e t h e d i e t a r y RNA c o n t e n t c a n be r e l a t i v e l y h i g h ; (c) t h e h i g h e n z y m e M e v e l s i n r u m i n a n t s e n s u r e t h a t r u m i n a l b a c t e r i a l RNA i s e f f e c t i v e l y h y d r o l y s e d and u t i l i z e d b y t h e h o s t t o s u p p l y a d d i t i o n a l n e c e s s a r y n i t r o g e n a n d p h o s p h a t e f o r i t s d i e t . R e a s o n s f o r s t u d y i n g v n u c l e a s e s T h e s e m i g h t be g r o u p e d i n t o f o u r c a t e g o r i e s : (a) t o c o r r e l a t e t h e enzyme a c t i v i t y w i t h t h e g e n e r a l s t a t e o f t h e o r g a n i s m e . g . i n r e g e n e r a t i n g l i v e r o r i n t h e m a t u r a t i o n o f i n t e s t i n a l e p i t h e l i a l c e l l s a s t h e y m i g r a t e f r o m c r y p t t o v i l l u s t i p ; (b) t o i n v e s t i g a t e t h e c y t o c h e m i s t r y o f t h e enzyme w i t h a v i e w t o g a i n i n g a n u n d e r s t a n d i n g o f i t s c e l l u l a r d i s t r i b u t i o n , f u n c t i o n a n d r e g u l a t i o n ; (c) t o s t u d y i t s e n z y m o l o g y p e r s e ; (d) t o i s o l a t e t h e enzyme f o r u s e a s a t o o l i n t h e s t u d y o f t h e p r i m a r y s t r u c t u r e o f n u c l e i c a c i d s . 11 Of t h e s e , t h e g r e a t e s t d r i v e i n t h e p a s t 20 y e a r s has b een t o w a r d (e.) and p a r t i c u l a r l y (d) . I n some c a s e s t h i s has been v e r y s u c c e s s f u l . The p i o n e e r i n g work o f H o l l e y e t a l . (1965) i n t h e s t r u c t u r a l d e t e r m i n a t i o n o f t h e c o m p l e t e s e q u e n c e o f y e a s t a l a n y l tRNA w o u l d have been v i r t u a l l y i m p o s s i b l e w i t h o u t t h e a v a i l a b i l i t y o f s p e c i f i c r i b o n u c l e a s e s . A n o t h e r example o f n u c l e a s e s employed a s t o o l s w o u l d be t h e o r i g i n a l ( F o s s e e t a l . , 1961) and p r e s e n t d a y ( S g a r a m e l l a & K h o r a n a , 1972) u s e o f PDase I I ( f r o m s p l e e n ) i n t h e c o n f i r m a t i o n o f d e o x y n u c l e o t i d e s e q u e n c e s by n e a r e s t n e i g h b o u r a n a l y s i s . The c l a s s i c a l s t u d i e s o f L e b l o n d & S t e v e n s (1948) have shown t h a t i n t e s t i n a l mucosa i s an a c t i v e l y p r o l i f e r -a t i n g t i s s u e w i t h a h i g h m i t o t i c a c t i v i t y . I t w o u l d t h u s a p p e a r t o be a u s e f u l t i s s u e i n w h i c h t o s t u d y c e r t a i n a s p e c t s o f n u c l e i c a c i d m e t a b o l i s m e . g . p o i n t s ( a ) a a n d (b) above m i g h t p r o f i t a b l y be i n v e s t i g a t e d w i t h t h i s s y s t e m . T h i s was t h e o r i g i n a l p u r p o s e o f t h e p r e s e n t s t u d y . P h o s p h o d i e s t e r a s e I I T h i s enzyme, w h i c h has b een p r i m a r i l y s t u d i e d i n mammalian t i s s u e s , i s a c t i v e a g a i n s t a v a r i e t y o f n a t u r a l and . s y n t h e t i c n u c l e o t i d e s u b s t r a t e s ( T a b l e I I I ) . E x t e n s i v e p u r i f i c a t i o n and c h a r a c t e r i z a t i o n o f t h e enzymes f r o m c a l f s p l e e n ( H i l m o e , 1960; R a z z e l l & K h o r a n a , 1 9 6 1 ) , hog s p l e e n 12 T a b l e I T T . Some s u b s t r a t e s f o r PDase I I 1 " S u b s t r a t e P r o d u c t m e a s u r e d ' R e f e r e n c e N a t u r a l ApU dTpdT, dTpdTp, dTpdTpdTpdT o l i g o r i b o -n u c l e o t i d e s " 3 o l i g o d e o x y r i b o -n u c l e o t i d e s ' 1 * Ap' 2, U'2 T p ? 2 a c i d s o l . u v a b s o r b i n g m a t e r i a l H i l m o e C1960) R a z z e l l & K h o r a n a (1961) H i l m o e ( I 9 6 0 ) B e r n a r d i & . ' B e r n a r d i (1968) C h r o m o g e n i c dTpNP dTpDNP b i s - ( p - n i t r o -p h e n y l ) p h o s p h a t e t h y m i d i n e 3'-( O - m e t h o x y f l u o r -e s c e i n ) p h o s p h a t e NP DNP NP O - m e t h o x y f l u o r e s c e i n R a z z e l l & K h o r a n a (1961) Menon & S m i t h (1970) B e r n a r d i & B e r n a r d i (1968) Z a r u b a e t a l . (1967) H i s t o c h e m i c a l t h y m i d i n e 3 * - ( a - r a - n a p h t h o l S i e r a k o w s k a naphthyl)phospEate- 5 e t a l . (1963) t h y m i d i n e 3'- ( 5 - 5 , 5 1 - d i b r o m o - 4 , 4 1 - W o l f e t a l . b r o m o - 4 - c h l o r o - dichloroindigo (1968I -3-i.ndolylI p h o s p h a t e T a b l e I I I ( c o n t . ) S u b s t r a t e P r o d u c t m e a s u r e d R e f e r e n c e O t h e r s a d e n o s i n e 3'- Ap'2 ( b e n z y l ) , p h o s p h a t e N - m e t h y l u r i d i n e N - m e t h y l Up 3 ( m e t h y l ) p h o s p h a t e t h y m i d i n e 3 ' - Tp* 2 X p h e n y l ) p h o s p h a t e Brown e t a l . (1954 H i l m o e (1960) Menon & S m i t h (1970) The l i s t i s by no means c o m p l e t e E s t i m a t e d by p a p e r c h r o m a t o g r a p h y The p r o d u c t s o f e x h a u s t i v e h y d r o l y s i s o f RNA b y p a n c r e a t i c RNAase. They c o n s i s t o f o l i g o n u c l e o t i d e s 3-5 r e s i d u e s l o n g (Markham & S m i t h , 1 9 5 2 ) . The p r o d u c t s o f h y d r o l y s i s o f DNA by s p l e e n DNAase I I . T h ey have an a v e r a g e s i z e o f 10-12 r e s i d u e s ( C a r r a r a & B e r n a r d i , 1 9 6 8 ) . T h i s compound was n o t c l e a v e d by s p l e e n PDase I I ( S i e r a k o w s k a e t a l . , 1 9 6 3 ) . The a - n a p h t h o l c a n be d i a z o t i z e d w i t h 5 - c h l o r o - O -t o l u i d i n e t o g i v e an i n s o l u b l e r e d dye ( S i e r a k o w s k a e t a l . , 1963). . 14 C B e r n a r d i & & B e r n a r d i , 1 9 6 8 ) s a n d salmon t e s t i s (Menon & S m i t h , 1970) has b e en c a r r i e d o u t . An a n a l o g o u s a c t i v i t y i n L a c t o b a c i l l u s a c i d o p h i l u s h a s a l s o b e en s t u d i e d by F i e r s & K h o r a n a ( 1 9 6 3 ) . PDase I I shows v i r t u a l l y no s p e c i f i c i t y t o w a r d t h e p u r i n e o r p y r i m i d i n e b a s e s o r t o w a r d t h e s u g a r m o i e t y o f t h e n u c l e o t i d e s u b s t r a t e s ' ( R a z z e l l & K h o r a n a , 1 9 6 1 ) . I n t h i s r e s p e c t , i t i s i n t e r -e s t i n g t h a t s u c h an a c t i v i t y has n o t b e en f o u n d i n E . c o l i , an o r g a n i s m whose complement o f n u c l e a s e s has b e en e x t e n s i v e l y s t u d i e d ( U c h i d a & E g a m i , 1971; Lehman, 1 9 7 1 ) . A c o n c l u s i o n w h i c h m i g h t be drawn f r o m t h i s i s t h a t PDase I I d o e s n o t p l a y a n i i m p o r t a n t r o l e i n s u c h f u n d a m e n t a l l y i m p o r t a n t p r o c e s s e s a s DNA s y n t h e s i s a n d / o r r e p l i c a t i o n . As men-t i o n e d p r e v i o u s l y s u c h a f u n c t i o n h a s been s u g g e s t e d f o r some n u c l e a s e s . Measurement o f p h o s p h o d i e s t e r a s e I I a c t i v i t y As Swenson & Hodes (1969) have p o i n t e d o u t , t h e a s s a y o f PDase i s c o m p l i c a t e d by t h e p r e s e n c e o f a number o f enzymes o f sometimes o v e r l a p p i n g a c t i v i t y a b l e t o h y d r o l y s e d o u b l y e s t e r i f i e d o r t h o p h o s p h o r i c a c i d . I n f a c t a d i s p u t e c u r r e n t l y e x i s t s as t o w h i c h i s t h e s u b s t r a t e o f c h o i c e f o r t h e d e t e r m i n a t i o n o f PDase I I ( R a z z e l l , 1967; B e r n a r d i & B e r n a r d i , 1 9 6 8 ) . S i n c e t h e s y n t h e t i c p h o s p h o -d i e s t e r t h y m i d i n e 3 1 - ( p - n i t r o p h e n y l ) p h o s p h a t e , i n t r o d u c e d 15 o r i g i n a l l y by Razzell & Khorana (1961) i s hydrolysed to a small degree by "homogeneous" DNAase II from spleen, (Bernardi & G r i f f e , 1964), Bernardi & Bernardi (1968) have claimed that thisccompound has serious drawbacks as a PDase II substrate. These workers prefer to use an exhaustive DNAase hydrolysate (Table III) despite the fact that Swenson & Hodes (1969), Sior (1970) and Sior & Hodes (1970) have convincingly shown that the hydrolytic a c t i v i t y displayed by p u r i f i e d hog spleen DNAase toward thymidine 3'-(p-nitrophenyl) phosphate (Bernardi & G r i f f e , 1964) was in fact due to contamination of the preparation by a non-s p e c i f i c PDase (see Part @ of the Results and Discussion section) . Evidence-in this'.thesis w i l l also sKo.w.thafefheir viewpoint i s unnecessarily pessimistic and that such chromogenic substrates as thymidine 3'-(p-nitrophenyl) phosphate and thymidine 3'-(2,4-dinitrophenyl) phosphate can be used i n the unambiguous and convenient measurement of PDase II a c t i v i t y . In p a r t i c u l a r , the dinitrophenyl ester (von Tigerstrom & Smith, 1969; Menon & Smith, 1970) has been found to be very useful for a number of reasons. The most important of these (von Tigerstrom & Smith, 1969) re l a t e s to the fact that since the yellow colour of DNP (pK=4.0) i s f u l l y developed at pH 6-7 (the optimum for PDase II a c t i v i t y ) , the use of t h i s substrate enables the a c t i v i t y of PDase II to be measured d i r e c t l y and continuously i n a^spectrophoto-meter cuvette. This substrate i s also hydrolysed by PDase 16 I I 1.4 - 1.9 t i m e s f a s t e r t h a n dTpNP (von T i g e r s t r o m & S m i t h , 1969; Menon & S m i t h , 1970; a l s o t h e p r e s e n t w o r k ) , t h u s i n c r e a s i n g t h e s e n s i t i v i t y o f t h e a s s a y . A f u r t h e r p r a c t i c a l c o n v e n i e n c e i s t h a t s i n c e t h e a b s o r p t i o n maximum o f DNP i s a t 3 60 nm t h e p r o d u c t may be e s t i m a t e d b y u s i n g t h e s p e c t r o p h o t o m e t e r i n e i t h e r t h e u v o r v i s i b l e modes. F i n a l l y t h e p r e s e n t w o r k h a s shown t h a t s i n c e t h e K m v a l u e f o r dTpDNP i s one t e n t h t h a t f o r dTpNP, much l e s s o f t h e d i n i t r o p h e n y l e s t e r n e e d b e u s e d i n t h e a s s a y f o r PDase I I w i t h a c o n s e q u e n t s a v i n g o f v a l u a b l e s u b s t r a t e . I n t e s t i n a l p h o s p h o d i e s t e r a s e s K h o r a n a (1961) h a s e m p h a s i z e d t h e h i s t o r i c a l i m p o r t -a n c e o f t h e u s e o f i n t e s t i n a l e x t r a c t s e s p e c i a l l y i n t h e o r i g i n a l i s o l a t i o n o f t h e d e o x y r i b o n u c l e o s i d e s ( L e v e n e & B a s s , 1931) a n d t h e d e o x y r i b o n u c l e o t i d e s ( K l e i n & T h a n n -h a u s e r , 1 9 3 5 ) . The e x i s t e n c e o f PDase I i n i n t e s t i n e was s u g g e s t e d b y t h e l a t t e r w o r k b u t i t was n o t u n t i l c o m p a r -a t i v e l y r e c e n t l y t h a t t h i s was c o n f i r m e d when H y n i e & Z b a r s k y ( 1970a,b) e x t e n s i v e l y p u r i f i e d and c h a r a c t e r i z e d t h e enzyme. T h e y showed t h a t t h e enzyme r e m a i n s i n t i m a t e l y a s s o c i a t e d w i t h a l k a l i n e p h o s p h o m o n o e s t e r a s e t h r o u g h much o f t h e c e l l f r a c t i o n a t i o n a n d t h e p u r i f i c a t i o n a n d , a n a l o g o u s l y , i s p r o b a b l y a c o n s t i t u e n t o f t h e b r u s h b o r d e r membrane. V e r y l a r g e amounts o f t h i s enzyme o c c u r i n i n t e s t i n e ( R a z z e l l , 1 9 6 1 ; Z a r u b a e t a l . , 1 967; H y n i e & Z b a r s k y , 1 9 7 0 a ) . 17 The o r i g i n a l d e m o n s t r a t i o n by Brown e t a l . (1954) o f t h e h y d r o l y s i s o f a d e n o s i n e 3 ' - b e n z y l p h o s p h a t e b y i n t e s t i n a l e x t r a c t s was t h e f i r s t i n d i c a t i o n t h a t a s e c o n d P Dase m i g h t be p r e s e n t i n t h e g u t . T h i s n o t i o n was c o n -f i r m e d b y H i l m o e a n d H e p p e l l ( 1 9 5 5 ) who, h o w e v e r , w e r e u n -s u c c e s s f u l i n p u r i f y i n g t h e enzyme b e y o n d a p r e l i m i n a r y s t a g e . The p r e s e n t i n v e s t i g a t i o n The s t i m u l u s f o r t h e p r e s e n t s t u d y was t h e o b s e r -v a t i o n b y H y n i e & Z b a r s k y (197 0a) t h a t a p p r o x i m a t e l y 60% o f t h e t o t a l P Dase I I a c t i v i t y i n h o m o g e n a t e s o f m u c o s a l s c r a p i n g s f r o m r a t i n t e s t i n e was f o u n d i n t h e s u p e r n a t a n t f r a c t i o n a f t e r u l t r a c e n t r i f u g a t i o n . T h i s o b s e r v a t i o n , w h i c h h a s b e e n r e p e a t e d ( F l a n a g a n , 1 9 7 0 ) , d i s a g r e e s w i t h f i n d i n g s o f o t h e r i n v e s t i g a t o r s ( v a n Dyck & W a t t i a u x , 1968; E r e c i n s k a e t a l ^ . , 1969) who h a v e r e p o r t e d t h a t PDase I I i s a l y s o s o m a l enzyme. A l t h o u g h t h e o b s e r v a t i o n s o f v a n D y c k & W a t t i a u x (1968) and E r e c i n s k a e t , a l . ( (1969) w e r e o b t a i n e d w i t h r a t l i v e r , one m i g h t e x p e c t a s i m i l a r d i s -t r i b u t i o n i n t h e s m a l l i n t e s t i n e a c c o r d i n g t o t h e w o r k o f Hsu & T a p p e l ( 1 9 6 4 ) . T h e s e a u t h o r s showed t h a t s i x m a r k e r enzymes f o r l y s o s o m e s w e r e s i m i l a r l y d i s t r i b u t e d among p a r -t i c u l a t e f r a c t i o n s f r o m l l i v e r a n d i n t e s t i n a l m u c o s a . I t seemed a p p r o p r i a t e t h e r e f o r e t o i n v e s t i g a t e more f u l l y t h e s u b c e l l u l a r l o c a t i o n o f PDase I I i n i n t e s t i n a l 18 mucosa. However, m u c o s a l s c r a p i n g s c a n c o n s i s t o f a m i x t u r e o f d i f f e r e n t c e l l t y p e s ( P o r t e o u s & C l a r k , 1965) w h i c h i s n o t s u r p r i s i n g i n v i e w o f t h e c o n s i d e r a b l e c o m p l e x i t y o f t h e i n t e s t i n a l mucosa as d e m o n s t r a t e d by t h e s c h e m a t i c d r a w i n g i n F i g . 1. So i n t h e p r e s e n t work p r e p a r a t i o n s o f e p i t h e l i a l c e l l s were u s e d b e c a u s e t h e s e c o n t a i n a r e l a t i v e l y Riomogeneous p o p u l a t i o n o f c e l l s (Evans e t a l . , 1 9 7 1 ) . E x p e r i m e n t s were c a r r i e d o u t w i t h i n t e s t i n a l p r e p a r a t i o n s f r o m t h e r a t r w h i c h c j c o n t a i n l a r g e amounts o f mucus and a r e somewhat d i f f i c u l t t o f r a c t i o n a t e by d i f f e r e n t i a l c e n t r i f u g a t i o n ( C l a r k & P o r t e o u s , 1965) and w i t h p r e p a r a t i o n s f r o m t h e g u i n e a p i g i n w h i c h t h e mucus c o n t e n t i s c o n s i d e r a b l y l o w e r ( H u b s c h e r e t a l . , 1965; E v a n s e t a l . , 1 9 7 1 ) . The work r e p o r t e d h e r e i n showed t h a t PDase I I f r o m g u i n e a p i g i n t e s t i n a l e p i t h e l i u m i s a l y s o s o m a l enzyme whereas t h e l o c a t i o n o f t h e r a t enzyme i s l e s s - c l e a r . D i f f e r e n t i a l c e n t r i f u g a t i o n o f i n t e s t i n a l homogenates f r o m b o t h a n i m a l s i n d i c a t e d t h a t g u i n e a p i g PDase I I was p r i m a r i l y a s s o c i a t e d w i t h t h e l y s o s o m a l f r a c t i o n wha3ea_s'tiietgrreat.er;partP o f t h e r a t enzyme a c t i v i t y a p p e a r e d i n t h e s u p e r n a t a n t f r a c t i o n r e m a i n i n g a f t e r u l t r a c e n t r i f u g a t i o n . M o r e o v e r t h e g u i n e a p i g enzyme a c t i v i t y d i s p l a y e d a s t r u c t u r e - l i n k e d l a t e n c y w h i c h was not;-apparent w i t h t h e r a t a c t i v i t y and was a l s o a s s o c i a t e d w i t h s u b c e l l u l a r p a r t i c l e s t h a t u n d e r w e n t a c h a r a c t e r i s t i c d e c r e a s e i n e q u i l i b r i u m d e n s i t y when T r i t o n 19 P e r i t o n e u m -Serosal aspect F i g . 1: A d i a g r a m a t i c r e p r e s e n t a t i o n o f t h e s m a l l i n t e s t i n e i n l o n g i t u d i n a l s e c t i o n . The d r a w i n g was t a k e n f r o m P o r t e o i u s (1972) . 20 WR-1339 was i n j e c t e d i n t o t h e a n i m a l s . B o t h enzyme a c t i v i t i e s w e r e p u r i f i e d b y c h r o m a t o -g r a p h y on DEAE c e l l u l o s e , C M - c e l l u l o s e a nd a g a r o s e . A much smaSer. d e g r e e o f p u r i f i c a t i o n was o b t a i n e d * , w i t h t h e g u i n e a p i g enzyme, a f i n d i n g w h i c h was p r o b a b l y r e l a t e d t o t h e t e n d e n c y o f t h e enzyme t o f o r m i n s o l u b l e a g g r e g a t e s u n d e r c e r t a i n c o n d i t i o n s . W i t h a f e w e x c e p t i o n s t h e p r o p e r t i e s o f t h e p a r t i a l l y p u r i f i e d P D a s e s w e r e r a t h e r s i m i l a r . A much more d e t a i l e d c h a r a c t e r i z a t i o n o f t h e r a t PDase I I was c a r r i e d o u t b e c a u s e o f i t s g r e a t e r p u r i t y . I t was f o u n d t o be an a c i d i c p r o t e i n o f m o l e c u l a r w e i g h t a b o u t 150,000 w h i c h d i d n o t c o n t a i n a n y c a r b o h y d r a t e . I t s h y d r o l y t i c p r o p e r t i e s i n d i c a t e d t h a t i t s s p e c i f i c i t y was s i m i l a r t o o t h e r b e t t e r - c h a r a c t e r i z e d P D a s e s o f t h i s t y p e . F i n a l l y a number o f e x p e r i m e n t s w i t h v a r i o u s enzyme ; i n h i b i t o r s w e r e c a r r i e d o u t . Of t h e s e , m o s t i n t e r e s t i n g w a s t t h e f i n d i n g t h a t i o d o a c e t i c a c i d i n a c t i v a t e d t h e enzyme, a p p a r e n t l y b y a l k y l a t i n g a n e s s e n t i a l r e s i d u e a t t h e a c t i v e c e n t r e o f t h e enzyme. 21 MATERIALS AND METHODS MATERIALS C h e m i c a l s G l u c o s e 6 - p h o s p h a t e ( d i s o d i u m s a l t ) , 2 - ( p - i o d o -p h e n y l ) - 3 - ( p - n i t r o p h e n y l ) - 5 - p h e n y l t e t r a z o l i u m c h l o r i d e , c y t o c h r o m e c , c a l f t hymus DNA, b i s - ( p - n i t r o p h e n y l ) p h o s p h a t e ( s o d i u m s a l t ) a n d p - c h l o r o m e r c u r i p h e n y l s u l p h o n i c a c i d ( s o d i u m s a l t ) w e r e p r o d u c t s o f t h e S i g m a C h e m i c a l Co., S t . L o u i s , Mo. Y e a s t RNA, RNA " c o r e " , p r o n a s e , p - c h l o r o m e r c u r i b e n z o a t e ( s o d i u m s a l t ) , p h o s p h o l i p a s e C ( C l o s t r i d i u m w e l c h i i ) a nd p h o s p h o l i p a s e D ( c a b b a g e ) w e r e f r o m C a l b i o c h e m , L a J o l l a , C a l i f . D i s o d i u m p - n i t r o p h e n y l p h o s p h a t e , t h y m i d i n e 3 ' - p h o s p h a t e ( b i s t r i e t h y l a m m o n i u m s a l t ) , a d e n y l y l - (3 ' ->5 ') - g u a n o s i n e , g u a n y l y l - (3 1 ->-5 1) - a d e n o s i n e , g u a n y l y l - (2 1->5 ') - a d e n o s i n e a n d d i c y c l o h e x y l c a r b o d i i m i d e w e r e p u r c h a s e d f r o m R a y l o C h e m i c a l s L t d . , E d m o n t o n , A l b e r t a . P o l y a d e n y l i c a c i d was f r o m ^ M i l e s o L a b o r a t o r i e s I n c . -K a n k a k e e , 1 1 1 . , anda-any o t h e r n u c l e o t i d e s w e r e p u r c h a s e d f r o m P-L B i o c h e m i c a l s I n c . , M i l w a u k e e , W i s . Whatman c h r o m a t o g r a p h y p r o d u c t s ( p a p e r s n o . 31 a n d n o . 4 0 and i o n e x c h a n g e c e l l u l o s e s DE22, DE32, DE52, CM22) w e r e b o u g h t f r o m M a n d e l S c i e n t i f i c Co., L t d . , M o n t r e a l , Quebec. S e p h a d e x G-100, G-150, G-150 s u p e r f i n e , S e p h a r o s e 6B, F i c o l l a n d ' B l u e D e x t r a n 2000 w e r e o b t a i n e d f r o m P h a r m a c i a (Canada) L t d . , D o r v a l , Quebec and B i o - G e l A-0.5m a n d B i o - G e l 22 HTP w e r e p u r c h a s e d f r o m B i o Rad L a b o r a t o r i e s , R i c h m o n d , C a l i f . S p l e e n p h o s p h o d i e s t e r a s e , E. c o l i a l k a l i n e p h o s p h a t a s e , c a t a l a s e , r i b o n u c l e a s e A, c h y m o t r y p s i n o g e n A, o v a l b u m i n a nd a l d o l a s e w e r e a l l W o r t h i n g t o n p r o d u c t s o b t a i n e d f r o m ICN C a n a d a L t d . , M o n t r e a l , Quebec. C o o m a s s i e B r i l l i a n t B l u e , m y o g l o b i n a nd a p o - f e r r i t i n w e r e p r o d u c t s o f S c h w a r z - M a n n , O r a n g e b u r g , New Y o r k . A c r y l a m i d e , m e t h y l e n e b i s - ( a c r y l a m i d e ) a n d N,N,N'^N' , t e t r a m e t h y l - e . t h y l e n e - d i a m i n e w e r e o b t a i n e d f r o m E a s t m a n Kodak Co., R o c h e s t e r , New Y o r k . LKB C a r r i e r A m p h o l y t e s ( A m p h o l i n e ) a n d Tween 80 w e r e b o u g h t f r o m The F i s h e r S c i e n t i f i c Co., V a n c o u v e r , B.C. T r i t o n X-100 was o b t a i n e d f r o m J . T . B a k e r , P h i l l i p s b u r g , New J e r s e y ; T r i t o n WR-1339 f r o m t h e R u g e r C h e m i c a l C o . , I r v i n g t o n - o n -H u d s o n , New Y o r k ; C r o t a l u s a d a m a n t e u s venom f r o m R o s s A l l e n ' s R e p t i l e I n s t i t u t e , S i l v e r S p r i n g s , F l o r i d a and b o v i n e s e r u m a l b u m i n ( f r a c t i o n V) f r o m t t h e A r m o u r P h a r m a c e u t i c a l C o ., C h i c a g o , 1 1 1 . O t h e r c h e m i c a l s w e r e t h e p u r e s t g r a d e a v a i l a b l e . D i p h e n y l a m i n e and o r c i n o l w e r e r e c r y s t a l l i z e d a s d e s c r i b e d b y S c h n e i d e r ( 1 9 5 7 ) . A n i m a l s R a t s ( W i s t a r ) w e i g h i n g a b o u t 200g e a c h , and a l b i n o g u i n e a p i g s o f t h e H a r t l e y s t r a i n (300-350g) w e r e o b t a i n e d f r o m t h e a n i m a l u n i t o f t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a and w e r e s t a r v e d £:,o>ri 24h p r i o r t o s a c r i f i c e . 23 METHODS DEAEC-chromatography For p u r i f i c a t i o n of the PDase II substrate, dTpDNP, Whatman DE22 was used. The anion-exchanger was precycled according to the manufacturers instructions i n 0.5M HCl and 0.5M NaOH and then equilibrated with 2M ammonium carbonate. Columns (generally 5cm x 30cm)1- of t h i s material were packed at 25°C under an applied a i r pressure of 5 p . s . i . and were then washed extensively with water u n t i l -.the effluent pH was 7 to 7.5. The water-soluble products of the synthetic reaction (see under Preparation and Characterization of thymidine 3 1 -(2,4-dinitrophenyl) phosphate) were applied to the column which was then washed with 1.! l i t e r of water followed by a four l i t e r s l i n e a r gradient which increased from OM to 0.1M with respect to triethylammonium bicarbonate. The l a t t e r was accomplished by placing two l i t e r s of water i n the mixing chamber and two l i t e r s of fi.lM triethylammonium bicarbonate in the s a l t chamber. Occasionally smaller columns were used and i n such cases the volume of the gradient was reduced propor-ti o n a t e l y . The uv (267nm) absorbing materials eluted from these columns were coll e c t e d and concentrated by f l a s h evaporation. Repeated addition of water and evaporation 'xThe dimensions given here (and hereafter! are those of the diameter x height of the packed chromatographic material. 24 e n s u r e d t h a t t h e t r i e t h y l a m m o n i u m b i c a r b o n a t e was c o m p l e t e l y r e m o v e d . The e n t i r e p r o c e d u r e w a s c c a r r i e d o u t , a s much a s p o s s i b l e , i n s u b d u e d l i g h t so a s t o m i n i m i z e t h e p h o t o ^ c a t a l y s e d h y d r o l y s i s o f t h e s u b s t r a t e ( von T i g e r s t r o m & S m i t h , 1 9 6 9 ) . F o r PDase I I p u r i f i c a t i o n Whatman DE32 o r DE52 was u s e d ; t h e r e a s o n f o r t h i s i s g i v e n i n P a r t B o f t h e R e s u l t s a n d D D i s c u s s i o n S e c t i o n . A f t e r p r e c y c l i n g , w h e r e n e c e s s a r y , a c c o r d i n g t o t h e m a n u f a c t u r e r ' s i n s t r u c t i o n s t h e DEAEC was e q u i l i b r a t e d w i t h and s t o r e d i n 0.5M NaH|POii t o w h i c h a few d r o p s o f t o l u e n e h a d b e e n a d d e d . PDase p u r i f i c a t i o n on DEAEC was g e n e r a l l y c a r r i e d o u t a t 4°C a t one o f two pH v a l u e s , e i t h e r pH 6.5 o r 7.6. When c h r o m a t o g r a p h y a t pH 6.5 was u s e d , a c o l u m n (2cm x 20cm) was p a c k e d u n d e r g r a v i t y w i t h t h e e x c h a n g e r a nd t h e n e q u i l i b r a t e d w i t h 20mM s o d i u m p h o s p h a t e b u f f e r pH 6.5. The enzyme s o l u t i o n was d i a l y s e d a g a i n s t t h i s B u f f e r o r a l t e r n a t i v e l y was d i l u t e d w i t h c o l d (4°C) d i s t i l l e d w a t e r u n t i l t h e c o n d u c t i v i t y o f t h e s o l u t i o n was e q u a l t o t h a t o f t h e c o l u m n e f f l u e n t b u f f e r . T hus p r e p a r e d , t h e p r o t e i n 1 s.'(©?lutpi'oin>n was a p p l i e d totthe;'DEAEC c o l u m n w h i c h was t h e n w a s h e d w i t h s t i a j r i t i i n g b u f f e r ( 0 . 5 - 1 . OX). More o f t e n , t h e p r o t e i n a d s o r p t i o n was c a r r i e d o u t u s i n g a " b a t c h " t e c h n i q u e . F o r t h i s t h e DEAEC w h i c h was e q u i l i b r a t e d a s u s u a l i n 2 0mM s o d i u m p h o s p h a t e b u f f e r pH 6.5, was a d d e d d i r e c t l y t o t h e p r o t e i n s o l u t i o n a n d t h e s u s p e n s i o n s t i r r e d 25 i g e n t l y f o r l h a t 4°C. The DEAEC (now w i t h p r o t e i n a d s o r b e d ) was c o l l e c t e d b y f i l t r a t i o n t h r o u g h a c o t t o n f i l t e r ( c u t f r o m a c l e a n l a b o r a t o r y c o a t ) a n d r e s u s p e n d e d i n t h e s t a r t i n g b u f f e r . Column p a c k i n g and w a s h i n g w e r e c a r r i e d o u t a s d e s c r i b e d a b o v e . The p r o t e i n s a d s o r b e d o n t h e s e c o l u m n s w e r e e l u t e d w i t h a l i n e a r g r a d i e n t composed o f 500ml o f 20mM s o d i u m p h o s p h a t e b u f f e r pH 6.5 and 500ml o f 0.15M s o d i u m p h o s p h a t e b u f f e r o f t h e same pH. When c h r o m a t o g r a p h y a t pH 7.6 was e m p l o y e d t h e same b a s i c t e c h n i q u e was u s e d w i t h t h e f o l l o w i n g e x c e p t i o n s : a l a r g e r c o l u m n o f DEAEC, 3.2cm x 28cm, was u s e d and t h e s t a r t i n g b u f f e r was 5mM T r i s - 5 m M NaH2POitpH 7.6. The p r o t e i n s w e r e e l u t e d f r o m t h e c o l u m n b y means o f a l i n e a r g r a d i e n t composed o f IZ o f s t a r t i n g b u f f e r w h i c h was made 1 M w i t h r e s p e c t t o N a C l . CMC c h r o m a t o g r a p h y Whatman CM22 a f t e r p r e c y c l i n g a c c o r d i n g t o t h e m a n u f a c t u r e r ' s i n s t r u c t i o n s i n 0.5M NaOH a n d 0.5M H C l , was s t o r e d i n 0.5M s o d i u m a c e t a t e b u f f e r pH 5 t o w h i c h a f e w d r o p s o f t o l u e n e h a d b e e n a d d e d a s a p r e s e r v a t i v e . C o lumns o f CMC (2cm x 20cm) w e r e e q u i l i b r a t e d a t 4°C i n 50mM s o d i u m a c e t a t e b u f f e r pH 5. The p r o t e i n s o l u t i o n , w h i c h c o m p r i s e d t h e PDase I I - r i c h f r a c t i o n s f r o m DEAEC c h r o m a -t o g r a p h y , was g e n e r a l l y n o t d i a l y s e d a g a i n s t t h e s t a r t i n g b u f f e r s i n c e i t was f o u n d t h a t t h e PDase a c t i v i t y was 26 a d s o r b e d v e r y s t r o n g l y t o t h e CMC once t h e pH o f t h e s o l u t i o n was l o w e r e d t o 5; t h e presence, o f s a l t s up t o an i o n i c s t r e n g t h o f 0.15 d i d n o t a f f e c t t h i s a d s o r p t i o n . A f t e r w a s h i n g t h e column w i t h 200-400ml o f s t a r t i n g b u f f e r t h e a d s o r b e d p r o t e i n s were e l u t e d w i t h a l i n e a r g r a d i e n t composed o f 500ml o f s t a r t i n g b u f f e r and 500ml o f s t a r t i n g b u f f e r w h i c h was made 0.5M w i t h r e s p e c t o f N a C l . O c c a s i o n -a l l y , a g r a d i e n t i n c r e a s i n g f r o m 0M t o 1M i n N a C l was u s e d . A g a r o s e c h r o m a t o g r a p h y T h i s c h r o m a t o g r a p h i c p r o c e d u r e , a l t h o u g h e m p l o y i n g m a t e r i a l s u s u a l l y u s e d i n g e l - f i l t r a t i o n c h r o m a t o g r a p h y , i s n o t b a s e d on t h e p r i n c i p l e c o f g e l e x c l u s i o n . I n s t e a d , s u b t l e i o n - e x c h a n g e e f f e c t s a r e p r o b a b l y i n v o l v e d , a s w i l l be d i s c u s s e d i n d e t a i l i n P a r t B o f t h e R e s u l t s and D i s c u s s i o n s e c t i o n . B i o - G e l A-0.5m(100-200 mesh) was u s e d i n a l l t h e e x p e r i m e n t s d e s c r i b e d h e r e i n , a l t h o u g h v e r y s i m i l a r r e s u l t s w e r e * o b t a i n e d , on a number o f o c c a s i o n s , w i t h S e p h a r o s e 6B. The a g a r o s e b e a d s were s t o r e d i n 50mM sodium a c e t a t e - 0 . 5 M N a C l - 0 . 0 2 % sodium a z i d e pH 5 and were r e t u r n e d t o t h i s s o l u t i o n a f t e r u s e . Columns o f t h e a g a r o s e (3cm x 15cm) were e q u i l i b r a t e d a t 4°C w i t h 50mM sodium acetate-50mM N a C l pH 5. The p r o t e i n s o l u t i o n a p p l i e d t o t h e s e columns was g e n e r a l l y t h e e a c t i v e f r a c t i o n f r o m CMC c h r o m a t o g r a p h y . The [NaCl] o f t h i s s o l u t i o n was r e d u c e d t o a b o u t 0.05M b y 27 t h e a d d i t i o n o f up t o f o u r - v o l u m e s o f c o l d (4°C) d i s t i l l e d w a t e r p r i o r t o i t s a p p l i c a t i o n t o t h e a g a r o s e c o l u m n . I t s h o u l d be m e n t i o n e d t h a t , u n l i k e g e l - f i l t r a t i o n , t h i s c h r o m a t o g r a p h y was u n a f f e c t e d b y t h e a p p l i e d v o l u m e o f s o l u t i o n . A l t e r n a t i v e l y t h e P D a s e - r i c h f r a c t i o n s w e r e c o n c e n t r a t e d b y u l t r a f i l t r a t i o n t o a b o u t 5 0 - 1 0 0 m l ; t h i s s m a l l v olume was t h e n d i a l y s e d a g a i n s t s e v e r a l c h a n g e s o f t h e s t a r t i n g b u f f e r b e f o r e i t was a p p l i e d t o t h e a g a r o s e c o l u m n . A f t e r w a s h i n g t h e c o l u m n w i t h s t a r t i n g b u f f e r ( a b o u t 200ml) t h e a d s o r b e d PDase I I a c t i v i t y was remov e d b y i n c r e a s i n g t h e [ N a d ] o f t h e e l u t i n g b u f f e r f r o m 50mM t o 0. 2M o r more. I t s h o u l d be e m p h a s i z e d t h a t t h e b i n d i n g o f i n t e s t i n a l PDase I I a c t i v i t y t o t h e s e a g a r o s e c o l u m n s was c r i t i c a l l y d e p e n d e n t on t h e i o n i c s t r e n g t h o f t h e b u f f e r u s e d . The enzyme was a p p a r -e n t l y s e l e c t i v e l y a d s o r b e d t o t h e a g a r o s e i n 50mM s o d i u m a c e t a t e b u f f e r pH 5 c o n t a i n i n g up t o 80mM N a C l ; r a i s i n g t h e N a C l h i g h e r t h a n t h i s c a u s e d some o f t h e PDase a c t i v i t y t o a p p e a r w i t h t h e n o n - a d s o r b e d p r o t e i n s i n t h e f l o w - t h r o u g h v o l u m e . D N A - c e l l u l o s e c h r o m a t o g r a p h y The D N A - c e l l u l o s e was a k i n d g i f t f r o m D r . P e t e r Can d i d o who p r e p a r e d i t a c c o r d i n g t o t h e p r o c e d u r e o f B a u t z & Dunn (1971) u s i n g c a l f thymus DNA and Whatman CF-11 28 c e l l u l o s e p o w d e r . The s l u r r y o f DNA and c e l l u l o s e was a l s o i r r a d i a t e d w i t h u v l i g h t as d e s c r i b e d b y L i t m a n (1968) i n o r d e r t o p r o d u c e a c r o s s - l i n k e d DNA t r a p p e d on t h e c e l l u l o s e m a t r i x . Columns (0.8cm x 5cm) o f t h e m a t e r i a l (0.4-0.5mg DNA/ml) w e r e p a c k e d u n d e r g r a v i t y a t 4°C a n d e q u i l i b r a t e d w i t h 5mM T r i s - 5 m M NaH 2PCy-10mM N a C l b u f f e r pH7.6. The p r o t e i n s o l u t i o n was d i a l y s e d a g a i n s t s e v e r a l c h a n g e s o f t h i s s t a r t i n g b u f f e r a n d a p p l i e d t o t h e c o l u m n w h i c h was t h e n w a s h e d w i t h s t a r t i n g b u f f e r (10ml) f o l l o w e d b y 2 0 m l p o r t i o n s o f t h e s t a r t i n g b u f f e r w h i c h c o n t a i n e d . t h e f o l l o w i n g s o d i u m c h l o r i d e c o n c e n t r a t i o n s : 20mM, 40mM, 60mM, lOOmM a n d 200mM. G e l f i l t r a t i o n c h r o m a t o g r a p h y The m a t e r i a l s u s e d w e r e S e p h a d e x G-150 ( p a r t i c l e s i z e 40-120um) ; S e p h a d e x G-150 s u p e r f i n e ( p a r t i c l e s i z e 10-40ym) and B i o - G e l A-0.5m (100-200 m e s h ) . The d r y S e p h a d e x b e a d s (10-15g) w e r e s u s p e n d e d i n 1.5 l i t e r s o f d i s t i l l e d w a t e r i n a two l i t e r E r l e n m e y e r f l a s k . T h i s was p l a c e d i n a b o i l i n g w a t e r b a t h f o r 6h t o f a c i l i t a t e t h e c o m p l e t e s w e l l i n g o f t h e g e l . Columns o f t h e g e l s w e r e p r e p a r e d a c c o r d i n g t o t h e r e c o m m e n d a t i o n s g i v e n i n t h e P h a r m a c i a b o o k l e t , " S e p h a d e x , g e l f i l t r a t i o n i n t h e o r y a nd p r a c t i c e " . A P h a r m a c i a K25/45 c o l u m n e q u i p p e d w i t h two f l o w - a d a p t o r s was u s e d i n t h e e x p e r i m e n t s and t h i s was 29 f i l l e d a t 4°C t o g i v e a g e l b e d o f 2.5cm x 33-35cm. The e l u t i o n b u f f e r v a r i e d f r o m e x p e r i m e n t t o e x p e r i m e n t as i n d i c a t e d i n t h e R e s u l t s and D i s c u s s i o n s e c t i o n and u p w a r d f l o w e l u t i o n was e m p l o y e d t h r o u g h o u t . P r o t e i n s o l u t i o n s (2ml) w e r e a p p l i e d t o t h e c o l u m n s f o l l o w e d by 2 0 m l i l o f 10% s u c r o s e a n d t h i s " c u s h i o n " was i n t u r n f o l l o w e d by 2 0 0 m l o f e l u t i n g b u f f e r . N o n - e n z y m i c p r o t e i n m o l e c u l a r w e i g h t m a r k e r s w e r e d i s s o l v e d i n t h e a p p r o p r i a t e e l u t i n g b u f f e r a t a c o n c e n t r a t i o n o f 2-5mg/ml and t h e e l u t i o n o f t h e s e was e s t i m a t e d by t h e i r a b s o r b a n c e a t 2 80nm. Much l e s s o f t h e m a r k e r enzymes was n e e d e d s i n c e t h e s e w e r e e s t i m a t e d by t h e i r e n z y m i c a c t i v i t i e s . The m o l e c u l a r w e i g h t s o f t h e s t a n d a r d s and PDase I I w e r e r e l a t e d t o t h e i r e l u t i o n p o s i t i o n s as c h a r a c t e r i z e d by e i t h e r t h e i r d i s t r i b -u t i o n c o e f f i c i e n t s ( K ) o r , more s i m p l y , b y t h e i r e l u t i o n av v o l u m e s (V ) . K i s d e f i n e d as V -V /V -V ( L a u r e n t e av e o/ t o & K i l l a n d e r , 196 4) . The v o i d v o l u m e (V ) o f e a c h ' o c o l u m n was d e t e r m i n e d u s i n g t h e e x c l u d e d s u b s t a n c e B l u e D e x t r a n 2000 and t h e t o t a l v o l u m e (V f c) by m e a s u r i n g t h e v o l u m e o f w a t e r n e e d e d t o c o m p l e t e l y d i s p l a c e t h e g e l b e d . F l o w r a t e s i n c o l u m n c h r o m a t o g r a p h y The f l o w r a t e s i n a l l t h e c o l u m n p r o c e d u r e s j u s t d e s c r i b e d w e r e r e g u l a t e d by a d j u s t m e n t o f t h e h y d r o s t a t i c p r e s s u r e h e a d a p p l i e d t o t h e c o l u m n . F o r c h r o m a t o g r a p h y 30 on DEAEC, CMC, a g a r o s e a n d D N A - c e l l u l o s e t h i s was v a r i e d b y 30 a n d 80cm o f w a t e r w h i c h r e s u l t e d i n f l o w r a t e s o f 5 0 - 7 0 m l / h i n m o s t c a s e s . I n t h e g e l f i l t r a t i o n e x p e r -i m e n t s a c o n s t a n t h y d r o s t a t i c p r e s s u r e o f 10cm H 2 0 was m a i n -t a i n e d b y t h e u s e o f a M a r i o t t e f l a s k ; t h e f l o w r a t e was 1 0 - 1 2 m l / h . U l t r a f i l t r a t i o n An A m i c o n c e l l ( m o d e l 402) e q u i p p e d w i t h e i t h e r a UM20E o r a PM30 D i a f l o m e m b r a n e w a s u s e d . The u l t r a -f i l t r a t i o n was a c c o m p l i s h e d b y a p p l y i n g a p r e s s u r e o f 40 p . s . i . t o t h e c e l l f r o m a c o m p r e s s e d n i t r o g e n c y l i n d e r . When n o t i n u s e t h e membranes were s t o r e d i n 10% e t h a n o l . The f l o w r a t e s o b t a i n a b l e w i t h t h e s e membranes w e r e o c c a s i o n a l l y r e d u c e d c o n s i d e r a b l y b y p r o t e i n m a t e r i a l c l o g g i n g t h e p o r e s . I n s u c h c a s e s a b r i e f (3-5min) w a s h i n g i n O i l M NaOH was s u f f i c i e n t t o r e s t o r e t h e f o r m e r r a p i d f l o w r a t e , a l t h o u g h t h i s p r o c e d u r e s h o u l d be u s e d w i t h c a u t i o n s i n c e , a f t e r t h i s t r e a t m e n t , an i n c r e a s e d " l e a k i n e s s " o f t h e membranes was o c c a s i o n a l l y o b s e r v e d . P a p e r c h r o m a t o g r a p h y o f n u c l e o t i d e s D e s c e n d i n g c h r o m a t o g r a p h y was c a r r i e d o u t u s i n g Whatman n o s . 31 and 40 p a p e r s . The s o l v e n t s e m p l o y e d w e r e : A, . i s o p r o p a n o l - a m m o n i a - w a t e r ( 7 : 1 : 2 , b y v o l . ) ; 31 B, n - b u t a n o l - a c e t i c a c i d - w a t e r ( 5 : 2 : 3 , b y v o l . ) ; C, e t h a n o l - l M ammonium a c e t a t e , pH 7 ( 7 : 3 ; v / v ) . N u c l e o t i d e s p o t s w e r e v i s u a l i z e d on t h e d r i e d c h r o m a t o g r a m s u n d e r uv l i g h t a n d whene n e c e s s a r y w e r e c u t o u t and e l u t e d f r o m t h e p a p e r b y t h e c e n t r i f u g a t i o n m e t h o d o f Markham as d e s c r i b e d b y H e p p e l (196 7 ) . I d e n t i f i c a t i o n o f 3 ' - n u c l e o t i d e s N u c l e o s i d e 3'- and 5 1 - p h o s p h a t e s e x h i b i t e d q u i t e s i m i l a r R f v a l u e s ' i n t h e s o l v e n t s y s t e m s named a b o v e . The 3 ' - r e s i d u e s h o w e v e r , c o u l d be r e a d i l y i d e n t i f i e d b y t h e i r r e s i s t a n c e t o t h e p o w e r f u l 5 1 - n u c l e o t i d a s e a c t i v i t y w h i c h i s p r e s e n t i n c r u d e s n a k e venom. I n c u b a t i o n o f 5'-n u c l e o t i d e s w i t h C r o t a l u s a damanteus venom (10yg/ml) i n 50mM t r i e t h y l a m m o n i u m b i c a r b o n a t e b u f f e r pH 8 o v e r n i g h t a t room t e m p e r a t u r e gave r i s e t o t h e c o r r e s p o n d i n g n u c l e o -s i d e s w h e r e a s 3 ' - n u c l e o t i d e s w e r e u n a f f e c t e d b y s u c h t r e a t m e n t . P r e p a r a t i o n and c h a r a c t e r i z a t i o n o f t h y m i d i n e 3 ' - ( 2 , 4 -d i n i t r o p h e n y 1 ) p h o s p h a t e The s y n t h e s i s was c a r r i e d o u t a c c o r d i n g t o t h e p r o c e d u r e o f v o n T i g e r s t r o m & S m i t h (1969) w i t h t h e f o l l o w -i n g m o d i f i c a t i o n s : ( i ) a t t h e e n d o f t h e r e a c t i o n p e r i o d t h e r e a c t i o n m i x t u r e was t a k e n up i n 150ml o f e t h e r and 32 e x t r a c t e d f o u r times w i t h 50ml p o r t i o n s of water; ( i i ) triethylammonium b i c a r b o n a t e was used to e l u t e products from the DEAEC column s i n c e i t could be removed e a s i l y and q u i c k l y from the e l u t e d substances by f l a s h evapor-a t i o n ; ( i i i ) no c a t i o n exchanger was added d u r i n g the c o n c e n t r a t i o n of the d e s i r e d product. As j u s t mentioned DEAEC chromatography was used to separate dTpDNP from the unreacted reagents. An example of such an experiment i s shown i n F i g . 2 . The s y n t h e t i c d i e s t e r was e l u t e d at a triethylammonium b i c a r b -onate c o n c e n t r a t i o n of about 0.04M immediately a f t e r a s m a l l peak of unreacted dTp and b e f o r e a l a r g e smear of DNP. The Rf values of dTp, dTpDNP and DNP i n s o l v e n t B were 0.39, 0.61 and 0.9 3 r e s p e c t i v e l y . The i d e n t i f i c a t i o n o f dTpDNP was confimed by the formation o f equimolar amounts of thymidine 3'-phosphate (whose i d e n t i t y was confirmed by the t e s t o u t l i n e d above) and DNP when the d i e s t e r was inc u b a t e d with spleen PDase. A l t e r n a t i v e l y , the f o l l o w i n g t e s t was c a r r i e d out. A s m a l l amount of dTpDNP (l-2mg) was d i s s o l v e d i n 1.1ml of water t o give s o l u t i o n A. To 1ml of s o l u t i o n A i n a 2ml v o l u m e t r i c f l a s k was added 0.1ml of 10M NaOH. Th i s mixture was incubated at 37°C f o r 5h and then d i l u t e d w i t h water to a volume of 2ml ( s o l u t i o n B) . S o l u t i o n s A and B were f u r t h e r d i l u t e d 100 f o l d and the s p e c t r a of these d i l u t e d s o l u t i o n s d e t e r -mined on a Cary 15 spectrophotometer; the r e s u l t s of such 33 T 1 1—; i i - i r FRACTION NO. F i g . 2: S e p a r a t i o n o f dTpDNP f r o m w a t e r - s o l u b l e r e a c t a n t s b y DEAEC c h r o m a t o g r a p h y . dTp (jO. 5 mmol) and DNP (10 mmol) w e r e r e a c t e d i n t h e p r e s e n c e o f DCC a s d e s c r i b e d b y v o n T i g e r s t r o m & S m i t h ( 1 9 6 9 ) . A n aqueous e x t r a c t o f t h e r e a c t i o n m i x t u r e was p r e p a r e d a n d c h r o m a t o g r a p h e d o n DEAEC a s d e s c r i b e d i n t h e M a t e r i a l s a n d M e t h o d s s e c t i o n . O t h e r d e t a i l s a r e g i v e n i n t h e t e x t . 34 an e x p e r i m e n t a r e shown i n F i g . 3. By u s i n g a v a l u e o f 13.9 x 10 3 l i t e r - m o l "^cm 1 f o r e3gQ and b y a s s u m i n g t h a t t h e c a l c u l a t e d .'j DNP i s e q u a l t o t h e o r i g i n a l dTpDNP i t i s p o s s i b l e t o c a l c u l a t e a v a l u e f o r e ? ? ? D N P f r o m t h e s e 26 5 d a t a . I n t h e e x p e r i m e n t d e s c r i b e d i n F i g . 3 a v a l u e o f 3 * -1 -1 20.9 x 10 l i t e r ^ m o l -cm was o b t a i n e d i n g o o d a g r e e m e n t w i t h t h e v a l u e s o f v o n T i g e r s t r o m & S m i t h (1969) and F l a n a g a n (19 70). B e c a u s e o f t h e i n h e r e n t i n s t a b i l i t y o f t h e d i e s t e r i n s o l u t i o n ( v o n T i g e r s t r o m & S m i t h , 1969) i t s l o w l y h y d r o l y s e d e v e n when k e p t a t 4°C and pH 4-6 i n t h e d a r k . T h e r e f o r e a t t i m e s i t was n e c e s s a r y t o r e p u r i f y t h e compound on a DEAE c e l l u l o s e c o l u m n . T h i s i s i m p o r t a n t s i n c e , as w i l l be shown l a t e r , one o f t h e p r o d u c t s o f h y d r o l y s i s , t h y m i d i n e 3 ' - p h o s p h a t e , i s a c o m p e t i t i v e i n h i b i t o r o f PDase I I . The i n s t a b i l i t y o f t h e n u c l e o t i d e was n o t o f s u c h m a g n i t u d e , h o w e v e r , t h a t i t i n t e r f e r e d w i t h t h e g e n e r a l a s s a y o f PDase I I a c t i v i t y . T a b l e I V shows t h e r a t e o f h y d r o l y s i s o f t h e compound i n a v a r i e t y o f b u f f e r s . Whereas a l o w d e g r e e o f b r e a k d o w n was o b s e r v e d a t pH v a l u e s o f 3, 6.1 and a t pH 9 i n T r i s - H C l b u f f e r , t h e r a t e o f 2 3 h y d r o l y s i s was f o u n d t o be 10 -10 t i m e s f a s t e r i n s e v e r a l g l y c i n e b u f f e r s a t pH 9. T h i s i n t e r e s t i n g r e s u l t was p r o b a b l y n o t t h e r e s u l t o f s i m p l e b a s e h y d r o l y s i s o f t h e d i e s t e r s i n c e t h e o b s e r v e d r a t e i n 0.1M NaOH, a l t h o u g h 35 i 1 1 1 1 I 1 1 1 1 1 r 2 4 0 280 320 360 4 0 0 440 480 nanometers F i g . 3: The s p e c t r a o f dTpDNP a n d i t s b a s e - c a t a l y s e d h y d r o l y s i s p r o d u c t s . A s o l u t i o n o f dTpDNP was p r e p a r e d ( s o l u t i o n A) and a p o r t i o n o f i t t r e a t e d w i t h I N NaOH a n d d i l u t e d t w o -f o l d ( s o l u t i o n B) as d e s c r i b e d i n t h e M a t e r i a l s a n d M e t h o d s s e c t i o n . 36 T a b l e IV. H y d r o l y s i s o f dTpDNP i n v a r i o u s b u f f e r s I n a volume o f l m l dTpDNP CO.57mMl was i n c u b a t e d a t 37 C w i t h t h e b u f f e r shown and t h e E^^Q o f t h e s o l u t i o n s was m e a s u r e d a t v a r i o u s t i m e s . B u f f e r PH l h . . 20h 0.1M g l y c i n e - H C l 3 <0.01 0.0.3 0.1M a c e t i c a c i d 3 <0.01 0.02 ;0.1M s o d i u m s s u c c i n a t e 1 6.1 <0.01 0. 08 0.1M T r i s - H C l 9 <0.01 -0.1M g l y c i n e - N a O H 9 1.08 -0.1M g l y c i n e - O . l M N a C l -NaOh 9 1.43 — 0.1M g l y c i n e 0.1M h y d r a z ine-NaOH 9 14.3 — 0.lM-NaOH i-12 0.96 PDase I I was a s s a y e d u n d e r t h e s e c o n d i t i o n s 37 q u i t e h i g h , was l o w e r t h a n t h a t f o u n d i n t h e g l y c i n e b u f f e r s j u s t m e n t i o n e d . A p o s s i b l e e x p l a n a t i o n m i g h t be d e d u c e d f r o m t h e w o r k o f K i r b y & V a r v o g l i s (196 8) who showed t h a t a w i d e v a r i e t y o f a m i n e s c a n h y d r o l y s e b o t h 2 , 4 - d i n i t r o -pheny\l p h o s p h a t e and b i s - (2 , 4 - d i n i t r o p h e n y 1) p h o s p h a t e . I n d e e d t h e r e s u l t s - i n T a b l e I V show some s t r i k i n g r e s e m b l e n c e s t o t h o s e o f K i r b y & V a r v o g l i s (196 8 ) . F o r i n s t a n c e , no h y d r o l y s i s o f 2 , 4 - d i n i t r o p h e n y 1 p h o s p h a t e was o b s e r v e d by t h e s e w o r k e r s i n t h e p r e s e n c e o f T r i s b a s e , w h e r e a s h y d r a z i n e was f o u n d t o c a u s e v e r y e x t e n s i v e h y d r o l y s i s o f t h e m o n e s t e r . F u r t h e r i n v e s t i g a t i o n o f t h e b r e a k d o w n o f dTpDNP i n g l y c i n e s o l u t i o n s a t pH 9 was n o t p o s s i b l e b e c a u s e o f t i m e l i m i t a t i o n s . P r e p a r a t i o n o f e p i t h e l i a l c e l l s u s p e n s i o n s The m e t h o d u s e d was e s s e n t i a l l y t h a t o f Connock & P o v e r ( 1 9 7 0 ) . G u i n e a p i g s a n d r a t s w e r e s t u n n e d b y b l o w s on t h e h e a d and k i l l e d b y d e c a p i t a t i o n . The s m a l l i n t e s t i n e o f e a c h a n i m a l was q u i c k l y r e m o v e d , c u t i n t o t h r e e o r f o u r p i e c e s f o r e a s e o f h a n d l i n g and c l e a n e d b y r e p e a t e d f l u s h i n g w i t h warm (37°C) 0.3M s u c r o s e - 5 m M s o d i u m p h o s p h a t e pH 7. F o l l o w i n g t h i s , one e n d o f e a c h p i e c e o f g u t was s e a l e d w i t h a s m a l l a r t e r y c l a m p , and t h e s e g m e n t f i l l e d w i t h warm (37°C) 1M urea-5n4M s o d i u m p h o s p h a t e pH 7 f r o m t h e o t h e r e n d w h i c h was t h e n s e a l e d w i t h a s e c o n d 38 c l a m p . The " s a c s " t h u s p r e p a r e d w e r e i n c u b a t e d a t 37°C i n 0.3M s u c r o s e - 5 m M s o d i u m p h o s p h a t e pH 7 f o r 15min a f t e r w h i c h t h e u r e a s o l u t i o n was d i s c a r d e d and r e p l a c e d w i t h i c e - c o l d 0.3M s u c r o s e - 5 m M s o d i u m p h o s p h a t e pH 7. S u b s e -q u e n t m a n i p u l a t i o n s w e r e c a r r i e d o u t a t 4°C. The " s a c s " w e r e now r o l l e d g e n t l y b e t w e e n thumb and f o r e f i n g e r , t h i s p r o c e d u r e b e i n g s u f f i c i e n t t o d i s l o d g e s h e e t s o f e p i t h e l i a l c e l l s f r o m t h e u n d e r l y i n g t i s s u e o f t h e i n t e s t i n a l m ucosa i n t o t h e s u c r o s e - p h o s p h a t e s o l u t i o n . The c e l l s u s p e n s i o n t h u s o b t a i n e d w e r e u s e d w i t h o u t f u r t h e r p u r i f i c a t i o n . A b o u t 2-3g o f c e l l s (wet w e i g h t ) w e r e o b t a i n e d f r o m e a c h r a t a n d 4-5g f r o m e a c h g u i n e a p i g . A s e r i e s o f p h o t o -m i c r o g r a p h s t a k e n a t v a r i o u s s t a g e s o f t h e p r o c e d u r e a r e shown i n F i g . 4. S u b c e l l u l a r f r a c t i o n a t i o n A l l o p e r a t i o n s w e r e c a r r i e d o u t a t 2-5°C. The c e l l s f r o m one a n i m a l , s u s p e n d e d i n 40ml ( a b o u t 8 v o l . ) o f 0.3M-s u c r o s e - 5 m M s o d i u m p h o s p h a t e , pH 7, w e r e h o m o g e n i z e d i n a P o t t e r - E l v e h j e m h o m o g e n i z e r w i t h 10 c o m p l e t e s t r o k e s o f t h e g l a s s v e s s e l a g a i n s t t h e T e f l o n p e s t l e , w h i c h was r o t a t i n g a t 900 r e v . / m i n . The homogenate was c e n t r i f u g e d i n a m o d e l PR-2 I n t e r n a t i o n a l c e n t r i f u g e ( r o t o r n o . 269) f o r l O m i n a t 400g and t h e t u r b i d s u p e r n a t a n t c a r e f u l l y r e m o v e d u s i n g a w i d e - t i p p e d P a s t e u r p i p e t t e a n d s a v e d . The l o o s e l y p a c k e d s e d i m e n t was r e s u s p e n d e d i n 40ml o f s u c r o s e - p h o s p h a t e F i g . 4: The i s o l a t i o n o f i n t e s t i n a l e p i t h e l i a l c e l l s . C e l l s u s -p e n s i o n s w e r e p r e p a r e d a s d e s c r i b e d i n t h e M a t e r i a l s and M e t h o d s s e c t i o n and s a m p l e s o f t h e p r e p a r a t i o n w e r e r e -moved a t v a r i o u s s t a g e s and e x a m i n e d by l i g h t m i c r o s c o p y . A, f r e s h l y i s o l a t e d u n t r e a t e d g u i n e a p i g i n t e s t i n e ( X 2 5 0 ) ; B, i n t e s t i n e a f t e r t r e a t m e n t w i t h IM urea-5mM s o d i u m p h o s p h a t e pH 7 ( X 2 5 0 ) ; C, t h e " c o r e " m a t e r i a l r e m a i n i n g a f t e r r e m o v a l o f t h e e p i t h e l i a l c e l l s ( X 2 5 0 ) ; D, E, F, s h e e t s o f e p i t h e l i a l c e l l s (D, E, X250; F, X400) . 40 medium and a g a i n h o m o g e n i z e d w i t h 5 s t r o k e s o f t h e homo-g e n i z e r . A f t e r c e n t r i f u g a t i o n a t 400g f o r l O m i n t h e s u p e r n a t a n t was r e t a i n e d and t h e s e d i m e n t washed t w i c e by d i s p e r s i o n i n 20ml o f s u c r o s e - p h o s p h a t e s o l u t i o n , g e n t l e h o m o g e n i z a t i o n (3 s t r o k e s o f t h e v e s s e l a g a i n s t t h e s t a t i o n a r y T e f l o n p e s t l e ) and c e n t r i f u g a t i o n a t 4 00g f o r l O m i n . The f i n a l s u p e r n a t a n t was o n l y s l i g h t l y c l o u d y . The w e l l - p a c k e d s e d i m e n t was s u s p e n d e d i n 4 0ml o f s u c r o s e -p h o s p h a t e t o g i v e f r a c t i o n I . The s u p e r n a t a n t s f r o m t h e f o u r c e n t r i f u g a t i o n s were combined and a 1ml sample removed f o r a n a l y s i s . F r a c t i o n s I-V were o b t a i n e d f r o m t h i s c y t o p l a s m i c " f r a c t i o n by d i f f e r e n t i a l c e n t r i f u g a t i o n . A t e a c h s t e p t h e s e d i m e n t was r e s u s p e n d e d i n 2 0ml o f s u c r o s e -p h o s p h a t e w h i l e t h e s u p e r n a t a n t was r e c e n t r i f u g e d a t t h e n e x t h i g h e s t s p e e d . The " c y t o p l a s m i c " f r a c t i o n was c e n t r i -f u g e d i n t h e I n t e r n a t i o n a l c e n t r i f u g e ( r o t o r no. 269) f o r l O m i n a t lOOOg t o y i e l d f r a c t i o n I I . F r a c t i o n s I I I and IV were o b t a i n e d u s i n g a S o r v a l l RC2-B c e n t r i f u g e e q u i p p e d w i t h an SS-34 r o t o r ; f r a c t i o n I I I by c e n t r i f u g a t i o n a t S'09Og f o r 10min,and f r a c t i o n IV by c e n t r i f u g a t i o n a t 9750g f o r 2 0min. The f i n a l c e n t r i f u g a t i o n was c a r r i e d o u t a t 105000g f o r 60min i n a S p i n c o no. 30 r o t o r t o g i v e f r a c t i o n V ( s e d i m e n t ) and f r a c t i o n IV ( s u p e r n a t a n t ) . I n some e x p e r i m e n t s t h e number o f c e n t r i f u g a t i o n s was r e d u c e d so t h a t f r a c t i o n s I + I I o r f r a c t i o n s I I + I I I + IV o r f r a c t i o n s I I I + IV o r f r a c t i o n s - I T T + IV + V were 41 i s o l a t e d t o g e t h e r . S o n i c a t i o n o f p a r t i c u l a t e f r a c t i o n s A m o d e l W-185-C B r a n s o n ? : s o n i f i e r was u s e d a t a power o u t p u t o f 100W ( 2 0 k H z ) . S u b c e l l u l a r f r a c t i o n s w e r e r e s u s -p e n d e d i n 2 0ml o f 5mM s o d i u m p h o s p h a t e , pH 6.5, and s o n i c a t e d i n a p o l y e t h y l e n e t u b e i m m e r s e d i n an i c e - w a t e r m i x t u r e by t h r e e 30s b u r s t s o f u l t r a s o u n d i n t e r s p e r s e d w i t h l m i n p e r i o d s o f c o o l i n g i n t h e i c e - w a t e r . The s u p e r n a t a n t o f t h i s e x t r a c t p r e p a r e d by c e n t r i f u g a t i o n f o r 60min a t 105000g was t h e s o n i c a t e d f r a c t i o n . D e n s i t y - g r a d i e n t c e n t r i f u g a t i o n o f p a r t i c u l a t e f r a c t i o n s I n e x p e r i m e n t s w h e r e t h i s p r o c e d u r e was u s e d f r a c t i o n s I I I + I V w e r e i s o l a t e d t o g e t h e r a n d r e s u s p e n d e d i n 0. 3M 5-sucrose-5mM s o d i u m p h o s p h a t e , pH 7 , as b e f o r e . P o r t i o n s (5ml) o f t h i s c o m b i n e d f r a c t i o n w e r e g e n t l y l a y e r e d on 30ml l i n e a r s u c r o s e g r a d i e n t s p r e p a r e d f r o m 20% (w/w) and 50% (w/w) s u c r o s e s o l u t i o n s . The c e n t r i f u g e t u b e s w e r e c e n t r i f u g e d f o r 3h a t 27,000 r e v / m i n i n a S p i n c o SW-27 r o t o r . A f t e r c e n t r i f u g a t i o n , t h e b o t t o m o f e a c h t u b e was p i e r c e d i n a Beckman r e c o v e r y s y s t e m and t h e c o n t e n t s r emoved f r o m t h e t o p b y s l o w l y p u m p i n g 60% (w/w) s u c r o s e t h r o u g h t h e h o l e . T en 3.5ml f r a c t i o n s w e r e o b t a i n e d f r o m e a c h g r a d i e n t . O c c a s i o n a l l y , t h e e x p e r i -m e n t a l p r o c e d u r e was v a r i e d s l i g h t l y and t h i s i s i n d i c a t e d , where n e c e s s a r y , i n t h e a p p r o p r i a t e f i g u r e l e g e n d . D e n s i t y - g r a d i e n t c e n t r i f u g a t i o n o f " s o l u b l i z e d " g u i n e a p i g and r a t PDase I I F r a c t i o n V I (see u n d e r S u b c e l l u l a r f r a c t i o n a t i o n ) was u s e d . P o r t i o n s o f t h i s f r a c t i o n (0.5ml) f r o m e a c h a n i m a l were p l a c e d on 1 2 . 5 m l l l i n e a r d e n s i t y g r a d i e n t s made f r o m 5% (w/w) and 2 0% (w/w) s u c r o s e s o l u t i o n s t h a t had be e n p r e p a r e d i n e i t h e r lOmM sodium p h o s p h a t e b u f f e r pH 6.5 o r i n lOmM sodium p h o s p h a t e - l M N a C l pH 6.5. A f t e r c e n t r i -f u g a t i o n l f o r 14h a t 39,OOOrev/min i n a S p i n c o SW-4 0 r o t o r t h e c o n t e n t s o f e a c h t u b e were removed a s d e s c r i b e d a b o v e . F o r t y f r a c t i o n s (5 f r o p s ) were o b t a i n e d f r o m e a c h g r a d i e n t . Ammonium s u l p h a t e f r a c t i o n a t i o n Two methods were employed. I n t h e f i r s t d i f f e r e n t amounts o f s o l i d (NHV) 2 S 0 4 were added t o 25ml p o r t i o n s o f a r a t h i g h - s p e e d s u p e r n a t a n t f r a c t i o n ( see u n d e r F r a c t i o n -a t i o n o f t h e c e l l s ) . The s u s p e n s i o n s were s t i r r e d f o r 30 m i n a t 4°C and c e n t r i f u g e d f o r an a d d i t i o n a l l O m i n a t 10,000 r e v / m i n i n t h e SS-34 r o t o r o f a S o r v a l l RC2-B c e n t r i -f u g e . P r e c i p i t a t e s were r e s u s p e n d e d and d i s s o l v e d i n 25ml o f 0.3M-sucrose-5mM-sodium p h o s p h a t e pH 7. In t h e s e c o n d method a s i n g l e 100ml p o r t i o n o f 43 h i g h - s p e e d s u p e r n a t a n t was u s e d , a nd t h e f r a c t i o n a t i o n was c a r r i e d b u t b y t h e a d d i t i o n o f s u c c e s s i v e a mounts o f ( N H i t ) 2 S 0 i f t o b t h i s s o l u t i o n . The t e c h n i q u e u s e d was s i m i l a r t o t h a t • i . e . e m p l o y e d i n t h e f i r s t m e t h o d e x c e p t t h a t a f t e r e a c h a d d i t i o n o f (NR\) 2S0ifthe p r e c i p a t e d p r o t e i n s w e r e r e m o v e d b y c e n t r i -f u g a t i o n b e f o r e t h e - a d d i t i o n s o f f u r t h e r s a l t t o t h e s u p e r -n a t a n t s o l u t i o n . I n e a c h c a s e t h e f o l l o w i n g f o r m u l a ( H e p p e l , 1955) was u s e d t o c a l c u l a t e t h e amounts o f ammonium s u l p h a t e a d d e d t o t h e s o l u t i o n s . 5 3 . 3 ( S 2 - S i ) x = 1 - 0 . 3 S 2 w h e r e x = g o f s o l i d (NH„) SO. t o be a d d e d t o 100ml o f a 4 2 4 s o l u t i o n o f s a t u r a t i o n S i t o n c h a n g e i t t o s a t u r a t i o n S 2 . S i a n d Sz a r e f r a c t i o n s o f s a t u r a t i o n ( p e r c e n t a g e s a t u r a t i o n / 100) a t 23°C. Zone p r e c i p i t a t i o n T h i s was c a r r i e d o u t i n t h e manner d e s c r i b e d b y P o r a t h (1962) o n a c o l u m n (3.4cm x 92cm) o f S e p h a d e x G-100 u s i n g ammonium s u l p h a t e a s t h e p r e c i p i t a t i n g a g e n t . E x p e r i m e n t s w e r e p e r f o r m e d u n d e r two s e t s o f c o n d i t i o n s . I n t h e f i r s t t h e c o l u m n was e q u i l i b r a t e d i n 50mM s o d i u m a c e t a t e - 3 M ammonium s u l p h a t e pH 5 and t h e e x p e r i m e n t was i n i t i a t e d b y a p p l y i n g t o • t h e c o l u m n a g r a d i e n t o f 44 ( N H i t J z S O i t w h i c h d e c r e a s e d f r o m 3M t o OM. T h i s was a c c o m p l i s h e d by p l a c i n g 150ml o f t h e s t a r t i n g b u f f e r i n t h e m i x i n g v e s s e l and 150ml o f 50mM sodium a c e t a t e pH 5 i n t h e r e s e r v o i r . When t h e e x p e r i m e n t was c a r r i e d o u t u n d e r t h e s e c o n d s e t o f c o n d i t i o n s t h e s t a r t i n g b u f f e r was 50mM sodium a c e t a t e - 1 . 5 M ammonium s u l p h a t e pH 5 and a 300ml g r a d i e n t o f ( N H O 2 S O 4 d e c r e a s i n g f r o m 1. 5M t o OM was u s e d . I n b o t h c a s e s , i m m e d i a t e l y a f t e r a p p l i c a t i o n o f t h e g r a d i e n t 30ml o f a h i g h - s p e e d s u p e r n a t a n t p r e p a r e d f r o m a r a t homogenate (see u n d e r S u b c e l l u l a r f r a c t i o n a t i o n ) was p l a c e d on t h e columns w h i c h were t h e n washed w i t h 50mM sodium a c e t a t e pH 5. E x t r a c t i o n o f r a t i n t e s t i n a l PDase I I N o r m a l l y t h e s t a r t i n g m a t e r i a l was an e p i t h e l i a l AS c e l l p r e p a r a t i o n , o b t a i n e d and d e s c r i b e d above. O c c a s i o n a l l y , however, t h e l a b o r i o u s d i s l o d g i n g o f t h e e p i t h e l i a l c e l l s was n o t c a r r i e d o u t . I n s t e a d t h e p i e c e s o f i n t e s t i n e were s c r a p e d w i t h a m i c r o s c o p e s l i d e t o g i v e a p r e p a r a t i o n o f " m ucosal s c r a p i n g s " . A l t h o u g h s l i g h t l y more p r o t e i n and PDase were o b t a i n e d f r o m t h e s e as compared w i t h t h e e p i t h e l i a l c e l l p r e p a r a t i o n s t h e r e s u l t s f r o m l b o t h were q u a l i t a t i v e l y v e r y s i m i l a r . A b o u t 4-6g o f m a t e r i a l were s u s p e n d e d i n 40ml o f 0.3M sucrose-5mM sodium p h o s p h a t e pH 7 and hom o g e n i z e d w i t h . 45 5 s t r o k e s o f t h e P o t t e r - E l v e h j e m h o m o g e n i z e r a s d e s c r i b e d e a r l i e r . The t i s s u e was c o m p l e t e l y p r o c e s s e d i n t h i s f a s h i o n , 10 r a t s y i e l d i n g a b o u t 200ml o f v i s c o u s h o m o g e n a t e . T h i s was p l a c e d i n t h e l a r g e v e s s e l o f a S o r v a l l O m n i - M i x e r and f u r t h e r h o m o g e n i z e d f o r 5min a t 8 00 r e v / m i n . A f t e r c e n t r i f u g a t i o n a t 9750g f o r l O m i n t h e s u p e r n a t a n t f r a c t i o n was s a v e d and t h e s e d i m e n t r e s u s p e n d e d i n 2 00ml o f f r e s h s u c r o s e - p h o s p h a t e s o l u t i o n . " The e n t i r e h o m o g e n i z a t i o n and c e n t r i f u g a t i o n p r o c e d u r e was r e p e a t e d on t h i s t o g i v e a s e c o n d e x t r a c t w h i c h was c o m b i n e d w i t h t h e f i r s t . O c c a s i o n a l l y , a t h i r d e x t r a c t i o n w i t h a n - a d d i t i o n a l 200ml o f s u c r o s e - p h o s p h a t e b u f f e r was c a r r i e d o u t i n t h i s way b u t n o r m a l l y t h i s was n o t d o n e . The c o m b i n e d e x t r a c t s w e r e t h e n f i n a l l y c e n t r i f u g e d a t h i g h - s p e e d ( 1 0 5 0 0 0 g f o r 60min) and t h e s u p e r n a t a n t f r a c t i o n f r o m t h i s was u s e d i n t h e e x p e r i m e n t s . E x t r a c t i o n o f g u i n e a p i g i n t e s t i n a l PDase I I F r a c t i o n s I I + I I I + I V , p r e p a r e d a s d e s c r i b e d u n d e r " S u b c e l l u l a r f r a c t i o n a t i o n " , w e r e u s e d a s t h e s t a r t i n g m a t e r i a l . The p a r t i c l e s w e r e r e s u s p e n d e d i n 10 v o l . o f 0.3M s u c r o s e - 5 m M - T r i s - 5 m M NaH2PO i * pH 7.6 a n d s o n i c a t e d a s d e s c r i b e d p r e v i o u s l y . O c c a s i o n a l l y , when l a r g e v o l u m e s o f m a t e r i a l had t o be p r o c e s s e d a B r o n w i l l s o n i c a t o r was u s e d . F i f t y m i l l i l i t e r p o r t i o n s o f t h e p a r t i c u l a t e s u s p e n s i o n s 46 were p l a c e d i n t h e w a t e r - c o o l e d v e s s e l o f t h i s i n s t r u m e n t and s o n i c a t e d f o r 5min a t 2 0kHz. The s o n i c a t e was c e n t r i f u g e d a t 9750g f o r 15min i n a S o r v a l l RC2-B c e n t r i f u g e e q u i p p e d w i t h a SS—34 r o t o r , and t h e s u p e r n a t a n t s a v e d . A f t e r r e s u s p e n s i o n o f t h e p e l l e t i n a volume o f f r e s h s u c r o s e - T r i s - p h o s p h a t e b u f f e r e q u a l t o t h a t o f t h e s u p e r n a t a n t , t h e m a t e r i a l was a g a i n s o n i c a t e d as b e f o r e . C e n t r i f u g a t i o n o f t h e s o n i c a t e gave a s e c o n d s u p e r n a t n a t e x t r a c t w h i c h was combined w i t h t h e f i r s t . I n most e x p e r i m e n t s t h e s e d i m e n t was r e - s o n i c a t e d f o r a t h i r d t i m e . I n t h i s way t h r e e e x t r a c t i o n s were p e r f o r m e d on t h e s u b -c e l l u l a r p a r t i c l e s and h i g h - s p e e d s u p e r n a t a n t p r e p a r e d f r o m t h e s e by u l t r a c e n t r i f u g a t i o n a t 105000g f o r 60min. I n some o f t h e e x p e r i m e n t s a 20mM T r i s - H C l b u f f e r o f pH 8 was u s e d i n s t e a d o f t h e s u c r o s e - T r i s - p h o s p h a t e b u f f e r d e s c r i b e d a b o v e . " A g i n g " o f p u r i f i e d r a t PDase I I P r e p a r a t i o n s o f PDase I I w h i c h had b een p u r i f i e d by c h r o m a t o g r a p h y on DEAEC, CMC, and a g a r o s e were d i a l y s e d a g a i n s t s e v e r a l c h a n g e s o f d i s t i l l e d w a t e r and l y o p h i l i z e d . The l y o p h i l i z e d powder was s t o r e d a t -2 0°C f o r p e r i o d s up t o 6 months w h i l e a d d i t i o n a l m a t e r i a l was b e i n g p r e p a r e d . S e v e r a l p r e p a r a t i o n s were combined and r e d i s s o l v e d i n 5mM T r i s - 5 m M NaH 2PCU pH 7.6 ( t h e m a t e r i a l was n o t v e r y s o l u b l e 47 i n s o d i u m a c e t a t e b u f f e r pH 5 ) , and t h e s o l u t i o n s o f o r m e d was k e p t a t 4°C f o r 3 weeks b e f o r e b e i n g u s e d i n t h e e x p e r i m e n t s . ' E l e c t r o f o c u s i n g An LKB 110ml a p p a r a t u s was u s e d b u t t h e m a n u f a c t -u r e r ' s p r o c e d u r e was m o d i f i e d s l i g h t l y s i n c e t h e recommended v o l u m e s w e r e f o u n d n o t t o c o m p l e t e l y f i l l t h e c o l u m n . The c o m p o s i t i o n s o f t h e s o l u t i o n s e m p l o y e d i n t h e p r e s e n t c a s e a r e g i v e n i n T a b l e V. The a n o d i c e l e c t r o d e s o l u t i o n was p l a c e d i n t h e a p p a r a t u s v i a t h e c e n t r a l t u b e w h i l e t h e s t a b i l i z i n g d e n s i t y g r a d i e n t was pumped ' s l o w l y i n t o t h e o u t e r t u b e by means o f a p e r i s t a l t i c pump. The g r a d i e n t was f o l l o w e d b y a n o v e r l a y s o l u t i o n w h i c h s e r v e d t o p r o t e c t t h e s a m p l e p r o t e i n s f r o m t h e h a r s h a l k a l i n e e n v i r o n m e n t o f t h e c a t h o d i c e l e c t r o d e s o l u t i o n w h i c h was p l a c e d on t h e c o l u m n l a s t . The w h o l e a p p a r a t u s was c o o l e d by p a s s i n g r u n n i n g w a t e r a t a t e m p e r a t u r e o f 5° -7°C t h r o u g h t h e o u t e r j a c k e t o f t h e c o l u m n . E x p e r i m e n t s w e r e r u n a t a c o n s t a n t v o l t a g e o f 300V f o r p e r i o d s o f t i m e b e t w e e n 24h and 90h and t h e i n i t i a l c u r r e n t was a b o u t 6mA d e c r e a s i n g o v e r t h e c o u r s e o f t h e e x p e r i m e n t t o o.8-0.4mA. Upon c o m p l e t i o n o f t h e e x p e r i m e n t , j u d g e d b y a l a c k o f f u r t h e r d e c r e a s e i n c u r r e n t , t h e a p p a r a t u s was e m p t i e d s l o w l y b y means o f a p e r i s t a l t i c pump. F r a c t i o n s (1.9ml)„ w e r e c o l l e c t e d a nd 48 T a b l e V. C o m p o s i t i o n o f t h e s o l u t i o n s -used i n t h e e l e c t r o f o c u s i n g e x p e r i m e n t s S u b s t a n c e A n o d i c s o l n . D e n s i t y g r a d i e n t O v e r l a y C a t h o d i c ( b o t t o m ) Dense s o l n . L i g h t * s o l n . s o l n . s o l n . ( t o p ) 10% Ampho-l i n e 2 s u c r o s e w a t e r c o n e . s u l p h -u r i c a c i d 12g 14.0ml 0.2ml 7.5ml 220ml 23g 0.5ml 26.5ml 47ml 1.5ml 10ml e t h a n o l a m i n e 0.2ml The p r o t e i n s a m p l e was a d d e d t o t h i s s o l u t i o n T h i s was p r e p a r e d b y d i l u t i n g 2.5ml o f t h e c o m m e r c i a l 4 0% s o l u t i o n t o 10ml w i t h H„0. 49 a s s a y e d f o r PDase I I a c t i v i t y ; t h e pH a t 4°C was a l s o m e a s u r e d i n t h e f r a c t i o n 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 The s y s t e m e m p l o y e d was a d a p t e d f r o m t h a t o f D a w L i s ( 1 9 6 4 ) . No s a m p l e o r s p a c e r g e l was u s e d a nd t h e s e p a r a t i n g g e l (0.5cm x 9.5cm) c o n s i s t e d o f 5.25% t o t a l a c r y l a m i d e (a 20:1 r a t i o o f a c r y l a m i d e t o m e t h y l e n e b i s -( a c r y l a m i d e ) was u s e d ) . The a c r y l a m i d e was d i s s o l v e d i n 0.37M Tris.-r-O'. 06M-H3C1 b u f f e r pH 8.7 w h i c h c o n t a i n e d 0.1% TEMED; o c c a s i o n a l l y u r e a was a d d e d t o a f i n a l c o n c e n t r a t i o n o f 4M. T h i s s o l u t i o n , i n a 100ml B u c h n e r f l a s k , was c o o l e d on c r a c k e d i c e and d e a e r a t e d u s i n g an a s p i r a t o r . F i n a l l y , t o i t j h e m i x t u r e was a d d e d s o l i d ammonium p e r s u l p h a t e (lmg/ml) and t h e s o l u t i o n was t h e n q u i c k l y p i p e t t e d i n t o s m a l l g l a s s t u b e s a n d c o v e r e d w i t h 0.2ml o f i s o b u t a n o l . P o l y m e r i z a t i o n was u s u a l l y c o m p l e t e w i t h i n 3 0min a f t e r w h i c h t h e i s o b u t a n o l was r e p l a c e d f i r s t w i t h d i s t i l l e d w a t e r and t h e n w i t h t h e e l e c t r o d e b u f f e r (5mM T r i s - 3 8 m M g l y c i n e pH 8 . 3 ) . The p r o t e i n s a m p l e w h i c h a l s o c o n t a i n e d 10% (w/v) s u c r o s e a n d 0.001% b r o m o p h e n o l b l u e was a p p l i e d u n d e r n e a t h t h i s s o l u t i o n i n a v o l u m e o f n o t g r e a t e r t h a n l O O u l . E l e c t r o p h o r e s i s was c a r r i e d o u t f o r 2'. 5-3h a t a c o n s t a n t c u r r e n t o f 1.5mA p e r t u b e . F o l l o w i n g t h i s , e a c h g e l w a s i d i s p l a c e d f r o m i t s t u b e b y p a s s i n g a s m a l l amount o f w a t e r b e t w e e n t h e g e l 50 and t h e i n s i d e s u r f a c e o f t h e t u b e by means o f a l o n g -t i p p e d s y r i n g e . D e t e c t i o n o f p r o t e i n i n p o l y a c r y l a m i d e g e l s T h i s was a c c o m p l i s h e d u s i n g a h o t s t a i n i n g method w i t h C o o m a s s i e B l u e (R.A.F. R e i t h m e i e r , p e r s o n a l commun-i c a t i o n ) . The g e l s were i n c u b a t e d a t 65°C f o r 30min i n 0.25% C o o m a s s i e B l u e - 4 5 % m e t h a n o l - 4 5 % w a t e r - 1 0 % a c e t i c a c i d . D e s t a i n i n g a l s o a t 65°C was c a r r i e d o u t by p l a c i n g t h e g e l s f i r s t i n 25% e t h a n o l - 6 5 % w a t e r - 1 0 % a c e t i c a c i d f o r l O m i n , t h e n f o r t h r e e a d d i t i o n a l p e r i o d s o f 20min e a c h i n f r e s h p o r t i o n s o f t h e same s o l u t i o n and f i n a l l y f o r 2 0min i n 10% a c e t i c a c i d . The r e m a i n i n g unbound s t a i n was removed by an o v e r n i g h t i n c u b a t i o n a t 24°C i n 10% a c e t i c a c i d and t h e d e s t a i n e d g e l s w e r e - a l s o s t o r e d i n t h i s ^ s o l u t i o n . D e t e c t i o n o f c a r b o h y d r a t e i n p o l y a c r y l a m i d e g e l s The PAS s t a i n a s d e s c r i b e d by Kapi-tany & Z e b r o w s k i (1973) was u s e d . D e t e c t i o n o f PDase I I a c t i v i t y i n p o l y a c r y l a m i d e g e l s Two methods were u s e d . I n t h e f i r s t t h e PDase I I a c t i v i t y was l o c a l i z e d i n s i t u by i n c u b a t i n g t h e g e l s a t 4 5°C i n t h e a s s a y medium f o r t h e enzyme. A y e l l o w - band 51 i n d i c a t e d t h e p r e s e n c e o f PDase a c t i v i t y . A l t e r n a t i v e l y t h e g e l was c u t i n t o 2mm o r 3mm s l i c e s u s i n g a d e v i c e s i m i l a r t o t h a t d e s c r i b e d by Chrambach ( 1 9 6 6 ) . E a c h p i e c e was f r o z e n (-20°C) and t h e n a l l o w e d t o s i t i n 5mM T r i s -5mM N a H a P O i t b u f f e r pH 7.6 (0.5ml) f o r 24h a t 4°C. P o r t i o n s o f t h e s e e x t r a c t s were t h e n a s s a y e d f o r PDase I I . L i g h t m i c r o s c o p y G u i n e a p i g i n t e s t i n a l t i s s u e (see u n d e r P r e p a r a t i o n o f e p i t h e l i a l c e l l s u s p e n s i o n s ) was k i n d l y p r e p a r e d f o r l i g h t m i c r o s c o p i c a l e x a m i n a t i o n l b y Mr. V i v i a n W y l i e u s i n g f a c i l i t i e s g e n e r o u s l y c o n t r i b u t e d by D r . M. H o l l e n b e r g i s l a b o r a t o r y i n t h e D e p a r t m e n t o f Anatomy. The i n t e s t i n e was s l i t o p e n , s p r e a d o u t on a f l a t s u r f a c e and 2mm2 p i e c e s were removed f r o m t h e a p p r o p r i a t e s e c t i o n s u s i n g a r a z o r b l a d e and p l a c e d i n a g l u t a r a l d e h y d e - f o r m a l d e h y d e f i x a t i v e s o l u t i o n w h i c h was a h a l f - s t r e n g t h v e r s i o n o f t h a t d e s c r i b e d b y K a r n o v s k y ( 1 9 6 5 ) . Clumps o f d i s l o d g e d e p i -t h e l i a l c e l l s were a l s o f i x e d i n t h i s s o l u t i o n . A f t e r a f i x a t i o n p e r i o d o f 3h t h e t i s s u e was p o s t f i x e d f o r 3h i n 1% O s O i t i n 0.2M c a c o d y l a t e b u f f e r pH 7.4 and s t o r e d i n a c o l d s o l u t i o n o f 0.25M- s u c r o s e - 0 . I M c a c o d y l a t e b u f f e r pH 7.4. Samples were d e h y d r a t e d by p a s s a g e t h r o u g h 30%, 50%, 70%, 80%, 95% and a b s o l u t e e t h a n o l s o l u t i o n s and f i n a l l y i n p r o p y l e n e o x i d e . A f t e r o v e r n i g h t r o t a t i o n i n a h a l f j . a n d h a l f m i x t u r e o f p r o p y l e n e o x i d e / E p o n - A r a l d i t e ( L u f t , 19617 52 t h e s e c t i o n s w e r e vacuum i m p r e g n a t e d i n E p o n - A r a l d i t e f o r 5h a n d f i n a l l y embedded i n f r e s h p o r t i o n s o f t h i s m a t e r i a l . T h i c k s e c t i o n s (lum) w e r e c u t on a P o r t e r - B l u m m i c r o -tome,- s t a i n e d i n 1% t o l u i d i n e B l u e a n d e x a m i n e d i n a L e i t z m i c r o s c o p e . E l e c t r o n m i c r o s c o p y T h i s p r o c e d u r e was k i n d l y c a r r i e d o u t f o r t h e a u t h o r by Ms. E s t h e r Lo o f t h e C a n a d a D e p a r t m e n t o f A g r i c u l t u r e R e s e a r c h S t a t i o n . The p a r t i c u l a t e s u s p e n s i o n s t o be e x a m i n e d w e r e d i l u t e d w i t h 5 v o l . o f 0.3M s u c r o s e - 5 m M s o d i u m p h o s p h a t e b u f f e r pH 7 a n d c e n t r i f u g e d a t 105000g f o r 6 0 m i n . The s e d i m e n t s s o f o r m e d w e r e f i x e d i n 5% g l u t a r a l d e h y d e i n O . l M - s o d i u m p h o s p h a t e b u f f e r pH 7.2 f o r l h , w a s h e d t w i c e , f o r 15min e a c h t i m e , i n t h e 0.1M s o d i u m p h o s p h a t e b u f f e r a b o v e , a nd t h e n p o s t f i x e d i n 0.028M a c e t a t e - v e r o n a l b u f f e r pH 7.2 ( P a l a d e , 1952) c o n t a i n i n g 1% OsOi+ f o r l h . The p e l l e t s w e r e t h e n d e h y d -r a t e d b y p a s s a g e t h r o u g h 7 0 % , 95% and a b s o l u t e e t h a n o l and f i n a l l y i n p r o p y l e n e o x i d e ( 1 0 - 1 5 m i n e a c h ) , i n f i l t r a t e d o v e r n i g h t i n a 1:1 m i x t u r e o f p r o p y l e n e o x i d e a n d - E p o n , embedded i n p u r e E p o n a n d c u r e d a t 60°C f o r 2 4 - 4 8 h . T h i n s e c t i o n s w e r e c u t o n a R e i c h a r t u l t r a m i c r o t o m e w i t h a d i a m o n d k n i f e , s t a i n e d i n i t i a l l y f o r 2 0min w i t h . 5% u r a n y l a c e t a t e c o n t a i n e d i n 50% MeOH, a n d t h e n f o r l O m i n i n t h e l e a d 53 c i t r a t e s o l u t i o n d e s c r i b e d by R e y n o l d s (1963) and f i n a l l y e xamined u s i n g a P h i l i p s EM 300 e l e c t r o n m i c r o s c o p e . Enzyme d e t e r m i n a t i o n s The a s s a y f o r PDase I I was c a r r i e d o u t i n 0.1M sodium s u c c i n a t e , pH 6.1, 0.1% (v/v) T r i t o n X-100 and 0.5mM t h y m i d i n e 3 1 - ( 2 , 4 - d i n i t r o p h e n y l ) p h o s p h a t e w i t h a s u i t a b l e d i l u t i o n o f enzyme. When s o l u b l e enzyme p r e -p a r a t i o n s were u s e d , t h e d e t e r g e n t was sometimes o m i t t e d . The r e l e a s e o f 2 , 4 - d i n i t r o p h e n o l was d e t e r m i n e d i n e i t h e r o f two ways: Method I was a c o n t i n u o u s a s s a y i n w h i c h l m l o f t h e r e a c t i o n m i x t u r e was p l a c e d i n a s p e c t r o p h o t o m e t e r c u v e t t e and t h e i n c r e a s i n g a b s o r b a n c e o f t h e s o l u t i o n a t 360nm was m e a s u r e d a t 37°C i n a G i l f o r d m o del 2000 s p e c t r o -p h o t o m e t e r . Method I I was a f i x e d - t i m e a s s a y w h i c h was c a r r i e d o u t i n a volume o f 0.3ml. A f t e r an i n c u b a t i o n o f 20min a t 37°C t h e r e a c t i o n was s t o p p e d w i t h 0.7ml o f 0.1M=-NaOH and t h e E _,»of t h e s o l u t i o n m e a s u r e d . C o n t r o l s —360 c o n t a i n e d t h e same components b u t t h e s u b s t r a t e was added a f t e r t h e NaOH. N o n e n z y m a t i c h y d r o l y s i s o f t h e s u b s t r a t e was n e g l i g i b l e u n d e r t h e s e a s s a y c o n d i t i o n s , as shown by t h e d a t a i n T a b l e IV. Whenever p o s s i b l e Method I was u s e d b e c a u s e o f t h e e a s e w i t h w h i c h i n i t i a l r e a c t i o n v e l o c i t i e s c o u l d be d e t e r m i n e d . I t s h o u l d be m e n t i o n e d h e r e t h a t o c c a s i o n a l l y when Method I I was u s e d , t h e s u b s t r a t e 54 c o n c e n t r a t i o n was n o t a l w a y s 0.5mM. F o r example, when l o c a t i n g PDase I I a c t i v i t y i n column e f f l u e n t s , c o n c e n t r a t -i o n s o f 0.05mM-0.2mM were r o u t i n e l y u s e d and t h e p e r i o d o f r e a c t i o n was n o t c r i t i c a l l y m e a s u r e d . F o r t h i s r e a s o n t h e amounts o f PDase I I a c t i v i t y i n t h e f i g u r e s showing chroma-t o g r a p h i c p u r i f i c a t i o n s o f t h e enzyme were g e n e r a l l y n o t q u a n t i t a t i v e . An a c c u r a t e e s t i m a t e o f t h e amount o f a c t i v i t y p r e s e n t was o b t a i n e d i n s t e a d by c a r r y i n g o u t an a s s a y on t h e combin e d a c t i v e f r a c t i o n s u s i n g Method I . A c i d p h o s p h a t a s e [EC 3.1.3.2.] a c t i v i t y was d e t e r m i n e d by m e a s u r i n g t h e f l u o r i d e - s e n s i t i v e (see H u b s c h e r & West, 1965) h y d r o l y s i s o f p - n i t r o p h e n y l p h o s p h a t e a t pH 5 and 3 7 ° C . R e a c t i o n m i x t u r e s (0.4ml) c o n t a i n e d 50mM sodium a c e t a t e b u f f e r , pH 5.0, 0.1% (v/v) T r i t o n X-100, 4mM EDTA, 5mM p - n i t r o p h e n y l p h o s p h a t e and enzyme. C o n t r o l t u b e s c o n t a i n e d , a d d i t i o n a l l y , 2mM KF. The r e a c t i o n was i n i t i a t e d by t h e a d d i t i o n o f s u b s t r a t e and s t o p p e d a f t e r 5-10min by a d d i n g 0.6ml o f 0.1M NaOH. The a b s o r b a n c e s o f t h e s o l -u t i o n s were m e a s u r e d a t 400nm. S u c c i n a t e d e h y d r o g e n a s e [EC 1.3.99.1] a c t i v i t y was d e t e r m i n e d a c c o r d i n g t o t h e method o f P e n n i n g t o n ( 1 9 6 1 ) , w i t h t h e m o d i f i c a t i o n s d e s c r i b e d by P o r t e o u s & C l a r k (.1965) . The a s s a y f o r g l u c o s e 6-phosphataseEtE0.3...33.>3*J 9]^was b a s e d on t h a t o f H u b s c h e r & West ( 1 9 6 5 ) . The r e a c t i o n m i x t u r e (0.3ml) c o n t a i n e d 0.1M MES b u f f e r , pH 6.5, 2mM KF, 4mM EDTA, 8mM g l u c o s e 6 - p h o s p h a t e and enzyme Cup t o lOOug 55 o f p r o t e i n ) . The r e a c t i o n was s t a r t e d b y t h e a d d i t i o n o f enzyme a n d s t o p p e d a f t e r 1 5 m i n . b y a d d i n g 0.7ml o f t h e a s c o r b a t e - m o l y b d a t e r e a g e n t d e s c r i b e d b y /Ames (1966) . A f t e r f u r t h e r i n c u b a t i o n f o r 20min a t 45°C t h e Eg20 °^ t ^ L e s o l u t i o n was m e a s u r e d . The c o n t r o l l a c k e d g l u c o s e 6 - p h o s p h a t e . The a s s a y medium f o r a l k a l i n e r p h o s p h a t a s e c o n t a i n e d 0.1M T r i s - H C l b u f f e r pH 9.5, 0.05mM N a C l , ImM M g C l 2 , ImM p - n i t r o p h e n y l p h o s p h a t e and enzyme i n a v o l u m e o f 0.3ml. The r e a c t i o n - w a s c a r r i e d o u t f o r 15min a t 37°C, _; s t o p p e d b y t h e a d d i t i o n o f 0.7ml o f 0.IM NaOH and t h e E ^ Q Q o f t h e r e s u l t i n g s o l u t i o n m e a s u r e d . A l c o h o l d e h y d r o g e n a s e and c a t a l a s e - w e r e d e t e r m i n e d b y p r o c e d u r e s d e s c r i b e d i n t h e W o r t h i n g t o n Enzyme M a n u a l . The i n c u b a t i o n m i x t u r e f o r 5 ' - n u c l e o t i d a s e c o n t a i n e d 0.1M g l y c i n e , lOmM M g C l 2 , 3mM a d e n o s i n e 5'-p h o s p h a t e and enzyme s o l u t i o n i n a f i n a l v o l u m e o f l m l a t pH 9.0. A f t e r a r e a c t i o n p e r i o d o f 15m i n 0.5ml of."..10% TCA was a d d e d a n d t h e s u s p e n s i o n was a l l o w e d t o s t a n d a t 4°C f o r 5 m i n . The l i q u i d was c l a r i f i e d b y c e n t r i f u g a t i o n and t h e r e s u l t i n g s u p e r n a t a n t a s s a y e d f o r P^. DNAase I was a s s a y e d i n a v o l u m e o f 1.25ml c o n t a i n i n g 0.1M MES b u f f e r pH 6.8, O.lmg DNA, 0.01M M n C l 2 and enzyme. A f t e r a 15 m i n i n c u b a t i o n p e r i o d t h e r e a c t i o n was s t o p p e d w i t h 0.25ml o f 1 2 % TCA. The c l o u d y s o l u t i o n s w e r e a l l o w e d t o s t a n d a t 0°C f o r l O m i n , c l a r i f i e d b y c e n t r i f u g a t i o n a n d t h e E o c n o f t h e r e s u l t i n g s u p e r n a t a n t s m e a s u r e d . 56 DNAase I I was d e t e r m i n e d i n a s i m i l a r f a s h i o n e x c e p t t h a t t h e a s s a y m i x t u r e c o n t a i n e d 0.1M s o d i u m a c e t a t e b u f f e r pH 5, 0.2mg DNA, 0.01M EDTA and enzyme. RNAase a nd a d e n o s i n e d e a m i n a s e a s s a y s w e r e p e r f o r m e d a s d e s c r i b e d b y Menon & S m i t h (197 0 ) . The g - v a l u e s f o r 2 , 4 - d i n i t r o p h e n o l , p - n i t r o p h e n o l a n d F f o r m a z a n u n d e r t h e e x p e r i m e n t a l c o n d i t i o n s u s e d w e r e 1 3 9 0 0 , 17600 ( F l a n a g a n , 1970) and 20100 ( P e n n i n g t o n , 1961) l i t e r . m o l "'".cm 1 r e s p e c t i v e l y . Enzyme u n i t s E x c e p t f o r DNAase a u n i t o f enzyme a c t i v i t y was d e f i n e d a s t h e amount w h i c h p r o d u c e d l u m o l o f p r o d u c t p e r h o u r a t 37°C. One u n i t o f DNAase, on t h e o t h e r h a n d , was t h e amount c a u s i n g t h e h y d r o l y s i s i n l m i n o f a c i d s o l u b l e o l i g o n u c l e o t i d e s h a v i n g an E^gQ o f 1.0. A n a l y t i c a l m e t h o d s P r o t e i n was d e t e r m i n e d b y t h e m e t h o d o f L o w r y et_ a l . (1951) u s i n g b o v i n e serum a l b u m i n as t h e s t a n d a r d . DNA and RNA w e r e e x t r a c t e d and e s t i m a t e d a s d e s c r i b e d b y S c h n e i d e r ( 1 9 5 7 ) ; t h e c o m p l e t e p r o c e d u r e was c a l i b r a t e d b y a p p l y i n g i t t o known amounts o f t h e n u c l e i c a c i d s . I n o r g a n i c p h o s p h a t e was d e t e r m i n e d b y t h e m e t h o d o f Ames U966) . 57 S u c r o s e c o n c e n t r a t i o n s were m e a s u r e d u s i n g an. Abbe 60 r e f r a c t o m e t e r . The d e n s i t i e s o f t h e s e s o l u t i o n s were t h e n d e t e r m i n e d f r o m t a b l e s i n t h e Handbook o f C h e m i s t r y and P h y s i c s ( C h e m i c a l Rubber Company P r e s s , 5 4 t h E d i t i o n ) . Measurement o f s a l t c o n c e n t r a t i o n s was c a r r i e d o u t u s i n g a R a d i o m e t e r C o n d u c t i v i t y M e t e r . The m e a s u r e d c o n d u c t i v i t y was r e l a t e d t o a s a l t c o n c e n t r a t i o n by t h e u s e o f s t a n d a r d c u r v e s p r e p a r e d f r o m s o l u t i o n s o f known c o n -c e n t r a t i o n . 58 RESULTS AND DISCUSSION PART AY THE SUBCELLULAR LOCATION OF INTESTINAL PDASE I I The s u b c e l l u l a r l o c a t i o n o f i n t e s t i n a l P Dase I I was i n v e s t i g a t e d u s i n g t h e c l a s s i c a l t e c h n i q u e s o f d i f f e r e n t i a l c e n t r i f u g a t i o n a s d e v e l o p e d and e m p l o y e d by s u c h w o r k e r s a s C l a u d e , Hogeboom and de Duve. The m a j o r a l t e r n a t i v e m e t h o d f o r l o c a l i z a t i o n o f e n z y m e s , t h a t o f c y t o l o g i c a l o r h i s t o l o g i c a l s t a i n i n g , was n o t u s e d i n t h e p r e s e n t s t u d y b e c a u s e t h e two PDase I I s u b s t r a t e s d e v e l o p e d f o r s u c h a p u r p o s e ( s e e T a b l e I I ) h a v e s e r i o u s s h o r t c o m i n g s . F o r e x a m p l e , S i e r a k o w s k a e t a l . (1963) s y n t h e s i z e d t h y m i d i n e 3 1 - ( a - n a p h t h y l ) p h o s p h a t e f o r t h i s p u r p o s e b u t f o u n d t h a t t h e compound was t o t a l l y r e s i s t a n t t o s p l e e n P D a se I I . An i n d i g o g e n i c s u b s t r a t e , t h y m i d i n e 3-'- ( 5 - b r o m o - 4 - c h l o r o - 3 - i n d o l y l ) p h o s p h a t e , was u s e d b y W o l f e t a l . (1968) who f o u n d , h o w e v e r , t h a t d i f f e r e n t a r e a s o f t h e v - c e l l w e r e s t a i n e d a t d i f f e r e n t pH v a l u e s t h u s r a i s i n g t h e p o s s i b i l i t y t h a t t h e compound m i g h t be h y d r o l y s e d b y s e v e r a l e n z y m e s . D i f f e r e n t i a l c e n t r i f u g a t i o n o f e p i t h e l i a l c e l l h o m o g e n a t e s F r a c t i o n a t i o n o o f c e l l p r e p a r a t i o n s f r o m g u i n e a p i g a n d r a t g a v e t h e r e s u l t s p r e s e n t e d i n T a b l e V I w h i c h , shows t h e d i s t r i b u t i o n p a t t e r n s o f P D a s e XT and a number " T a b l e V I . S u b c e l l u l a r d i s t r i b u t i o n of components i n homogenates o f e p i t h e l i a l c e l l s from g u i n e a p i g and r a t i n t e s t i n e The a b s o l u t e v a l u e s a r e the t o t a l amounts o b t a i n e d from the e p i t h e l i a l c e l l s o f one a n i m a l and a r e e x p r e s s e d i n mg f o r p r o t e i n , DNA and RNA and i n u n i t s f o r the enzymes. The v a l u e s shown were o b t a i n e d by a d d i n g the amounts p r e s e n t i n the sedirr.ont and s u p e r n a t a n t a f t e r tho i n i t i a l c e n t r i f u g a t i o n s t e p . The of the amounts o f the i n -d i v i d u a l f r a c t i o n s I - V I , e x p r e s s e d as a p e r c e n t a g e o f the a b s o l u t e v a l u e gave the r e c o v e r y o f each component; t h e v a l u e s f o r the s i x f r a c t i o n s have been n o r m a l i s e d so t h a t t h e t o t a l i n eachcaso was e q u a l t o 100. The rr.eans (i S.D.) are shown and the f i g u r e s i n p a r e n t h e s e s r e f e r t o the numbers o f e x p e r i m e n t s p e r f o r m e d . Component A b s o l u t e Recovery • V a l u e s (%) P e r c e n t a g e o f r e c o v e r e d cor.por.ent i n e a c h f r a c t i o n I I I I I I IV V v : P r c t e i r . (4) 450 x 57 102. .0 z 6.7 21.3 t 3.9 9.8 i 2.1 9. .4 1 1 .0 9. 1 I 1.9 11, .1 t 0.2 39.4 i 0. .6 PDase I I (6) 226 t 47 95. .9 t 8.1 10.0 x 3.0 15.3 ± 2.3 24. .8 1 1 .0 18. 0 ± 2.7 11.9 t 1.1 20.0 i i . 1 ^.cii phoa. (5)' 424 t 72 101. .5 ± 3.9 16.6 t 7.7 8.7 1 2.6 25. .2 ± 3 .7 23. 2 X 3.2 17, .2 i 1.5 9.1 r 3. .2 G u i n e a CtU (4) 37.4 i 7.8 92. ,3 1 10.3 51.3 t 9.4 • 34.8 1 7.8 9. .1 i 2 .3 2. 0 1 0.9 1. . 6 t 0.1 1.1 x 2. .3 Pis EHA (S) 38.3 i 8.3 94. .3 1 8.5 20.0 t 6.7 8.6 t 1.9 6. .9 1 1 .9 • - 8. 8 1 1.0 30. .8 l 2.8 24.9 t 4. .1 SDH 411 t 139 88. ,0 + 10.1 15.9 t 10.2 5.7 i 1.4 42, .2 t 9 .4 28. 2 X 1.2 4. .2 x 1.0- 3.8 t 3. .3 C-6-Pace (4) 555 x 184 87, t 4.1 27.6 i 13.8 6.6 X 2.1 9, .4 x 4 .1 19. 7 t 6.3 29. .0 x 3.9 7.7 X <• .7 P r o t e i n (5) 273 t 53 100.9 t 2.7 34.2 t 2.4 6.0 I 0.7 7. .0 t 0.6 5. 2 t 0.4 10. , 4 X 1.82 37.2 i i . . 3 PDase I I [4) 116 t 45 90.6 - 3.5 16.4 t 2.5 10.1 ± 2.4 17. .5 i i .1 9. 2 x • 1.6 ' 7. ,9 X 1.7 39.0 X 5. .7 A£id phos. (5) 153 1 40 100, .4 t 9.0 35.1 i 6.4 6.0 t 2.0 18.1 t 5 .6 ' 13. 5 I 2.0 15. .6 t 3.5 11.6 t 3. .1 60 o f m a r k e r s f o r s u b c e l l u l a r c o m p e n e n t s : DNA f o r n u c l e i , s u c c i n a t e d e h y d r o g e n a s e f o r m i t o c h o n d r i a , a c i d p h o s p h a t a s e f o r l y s o s o m e s , RNA f o r r i b o s o m a l p a r t i c l e s and g l u c o s e 6-p h o s p h a t a s e f o r ^ e n d o p l a s m i c r e t i c u l u m . The r e l a t i v e s p e c i f i c a c t i v i t i e s o r amounts o f t h e s e c o m p o n e n t s a r e shown i n F i g . 5. P r e l i m i n a r y e x a m i n a t i o n b y l i g h t m i c r o s c o p y r e v e a l e d t h a t g r e a t e r amounts o f a m u c u s - l i k e amorphous m a t e r i a l w e r e p r e s e n t i n f r a c t i o n I o f t h e r a t t h a n t h a t f r o m t h e g u i n e a p i g . T h i s o b s e r v a t i o n c o u l d e x p l a i n t h e d i f f e r e n c e s f o u n d i n t h e r e l a t i v e amounts o f p r o t e i n r e c o v e r e d i n f r a c t i o n s I-V f r o m b o t h a n i m a l s ( T a b l e V I ) , s i n c e t h e mucus c o u l d t r a p some o f t h e s l o w e r -s e d i m e n t i n g p a r t i c l e s i n t h e l o w - s p e e d s e d i m e n t ( f r a c t i o n I ) . The l a r g e amount o f mucus i n r a t i n t e s t i n a l p r e p a r a t i o n s h a s b e e n shown i n p r e v i o u s i n v e s t i g a t i o n s t o impede • f r a c t i o n a t i o n o f t h e t i s s u e ( R o b i n s o n , 1 9 6 3 ; C l a r k & P o r t e o u s , 1 9 6 5 ) . M i c r o s c o p i c e x a m i n a t i o n a l s o showed t h a t b e s i d e s '...numerous n u c l e i , f r a c t i o n I c o n t a i n e d many u n b r o k e n c e l l s w h i l e f r a c t i o n I I was m o s t l y n u c l e a r . T h e r e f o r e s i n c e a s l i g h t l y b e t t e r s e p a r a t i o n o f t h e p a r t i c u l a t e f r a c t i o n s was a c h i e v e d w i t h t h e g u i n e a p i g , a more e x t e n s i v e b i o c h e m i c a l c h a r a c t e r i z a t i o n o f t h e s e was c a r r i e d o u t a s i l l u s t r a t e d b y t h e r e s u l t s shown i n T a b l e V I and F i g . 5. C o n s i d e r a b l e c r o s s - c o n t a m i n a t i o n a mongst t h e f r a c t i o n s was a p p a r e n t e s p e c i a l l y i n t h e " m i t o c h o n d r i a l " f r a c t i o n s I I I and I V , w h i c h , c o n t a i n e d t h e 61 -i r c o o E o o 2 ! E 0 ••-u o U 2 °u 0) D_ 0 »/> <D > _o P D a s e A c i d P h o s . D N A G-6 -Pase 1_H S D H R N A • J r - i G 50 100 50 protein content-}/ 100 F i g . 5: L o c a l i z a t i o n o f l u l a r f r a c t i o n s of i n t e s t i n a l ep i n d i c a t e the r e l the components c of component/per VI. F r a c t i o n s I c o r d i n g to t h e i r p i g ; R, r a t . PDase I I and ot h e r components i n s u b c e l -o b t a i n e d by d i f f e r e n t i a l c e n t r i f u g a t i o n i t h e l i a l c e l l homogenates. The o r d i n a t e s a t i v e s p e c i f i c a c t i v i t i e s (or amounts) of a l c u l a t e d f o r each f r a c t i o n as per cent cent of p r o t e i n u s i n g the data i n Table -VI are d e p i c t e d from l e f t to r i g h t ac-p r o t e i n contents ( a b s c i s s a ) ; G, guinea 62 h i g h e s t s p e c i f i c a c t i v i t i e s o f b o t h s u c c i n a t e d e h y d r o g -e n a s e a n d a c i d p h o s p h a t a s e ( F i g . 5 ) . The r e s u l t s o f o t h e r w o r k e r s ( P o r t e o u s & C l a r k , 1 9 6 5 ; H u b s c h e r e t a l . , 1 9 6 5 ; W r i g g l e s w o r t h & P o v e r , 1966) a l s o i n d i c a t e t h a t m i t o c h o n d r i a and l y s o s o m e s f r o m i n t e s t i n a l p r e p a r a t i o n s t e n d t o s e d i m e n t a t s i m i l a r c e n t r i f u g a l f o r c e s . I n a n y e v e n t i t was n o t p o s s i b l e t o s e p a r a t e p a r t i c l e s c o n t a i n i n g s u c c i n a t e d e y d r o g e n a s e f r o m t h o s e c o n t a i n i n g a c i d p h o s -p h a t a s e b y d i f f e r e n t i a l c e n t r i f u g a t i o n . The d i s t r i b u t i o n o f g u i n e a p i g PDase I I ( F i g . 5) was s i m i l a r t o t h e p a t t e r n s shown by t h e m a r k e r s f o r m i t o c h o n d r i a a n d , l y s o s o m e s . A s i m i l a r i t y o f t h e d i s t -r i b u t i o n s o f PDase and a c i d p h o s p h a t a s e b e t w e e n t h e r a t and g u i n e a p i g was a l s o o b s e r v e d ( F i g . 5 ) . H o w e v e r , t h e r e w e r e i m p o r t a n t d i f f e r e n c e s . F o r i n s t a n c e , i n t h e g u i n e a p i g 60% o f t h e PDase a n d a c i d p h o s p h a t a s e a c t i v i t i e s w e r e r e c o v e r e d i n f r a c t i o n s I I - I V ( T a b l e s V I and V I I ) , w h e r e a s i n t h e r a t l e s s t h a n 4 0% o f t h e s e enzymes was p r e s e n t i n t h e s e f r a c t i o n s ( T a b l e V I ) . F u r t h e r , t h e f i n a l s u p e r n a t a n t f r o m t h e r a t t i s s u e c o n t a i n e d a much h i g h e r p r o p o r t i o n o f t h e t o t a l PDase t h a n t h e c o r r e s p o n d i n g f r a c t i o n f r o m t h e g u i n e a p i g ( T a b l e V I ) . S i n c e t h e r a t p r e p a r a t i o n s w e re more v i s c o u s b e c a u s e o f t h e i r h i g h e r mucus c o n t e n t , i t was t h o u g h t t h a t t h e l a r g e r s h e a r i n g f o r c e s w h i c h d e v e l o p d u r i n g h o m o g e n i z a t i o n m i g h t l e a d t o a g r e a t e r d i s r u p t i o n o f t h e s u b c e l l u l a r p a r t i c l e s . However, when T a b l e V I I . D i s t r i b u t i o n o f PDase I I , a c i d p h o s p h a t a s e and p r o t e i n i n homogenates o f g u i n e a p i g i n t e s t i n a l e p i t h e l i a l c e l l s p r e p a r e d i n m e d i a o f d i f f e r e n t v i s c o s i t y . A l e s s p r o t r a c t e d v e r s i o n o f t h e n o r m a l p r o c e d u r e was u s e d i n w h i c h o n l y t h r e e f r a c t i o n s were o b t a i n e d ; t h e f i r s t c o r r e s p o n d e d t o f r a c t i o n s I + I I , t h e s e c o n d t o f r a c t i o n s I I I + I V + V a n d t h e t h i r d t o f r a c t i o n V I o f T a b l e V I . I n (a) t h e h o m o g e n i z i n g medium was 0.3M sucrose-5mM sodium p h o s p h a t e , pH 7; i n (b) i t c o n t a i n e d , a d d i t i o n a l l y , 3% F i c o l l . I n e a c h c a s e t h e mean v a l u e s (*S.D.) f o r 4 e x p e r i m e n t s a r e shown. Component P e r c e n t a g e o f r e c o v e r e d component i n e a c h f r a c t i o n I + I I I I I + IV + V V I P r o t e i n 25.4 ± 7.6 32.5 + 5.3 42.0 + 2.1 (a) PDase I I 22.6 ± 7.6 57.6 + 6.6 19.9 + 3.0 A c i d p h o s p h a t a s e 27.3 ± 4.7 59.9 + 3.9 12.7 + 1.4 P r o t e i n 28.0 ± 7.3 25.9 + 6.5 46.2 + 2.7 Cb) PDase I I 20.6 ± 6.1 59.8 + 4.7 19.7 + 1.9. A c i d p h o s p h a t a s e 26.6 ±10.6 59.5 + 9.9 13.9 + 3.2 64 t h e v i s c o s i t y o f g u i n e a p i g h o m o g e n a t e s was a r t i f i c a l l y i n c r e a s e d t o a p p r o x i m a t e t h a t o f r a t p r e p a r a t i o n s b y t h e a d d i t i o n o f a s u i t a b l e amount o f F i c o l l , t h e r e was no d i f f e r e n c e i n t h e d i s t r i b u t i o n s o f a c i d p h o s p h a t a s e and PDase I I ( T a b l e V I I ) . A c t i v a t i o n o f a c i d p h o s p h a t a s e and PDase I I I f PDase I I i s a l y s o s o m a l enzyme i n t h e i n t e s t i n a l e p i t h e l i u m i t w o u l d be e x p e c t e d t o e x h i b i t t h e " s t r u c t u r e -l i n k e d l a t e n c y " common t o o t h e r a c i d h y d r o l a s e s (de Duve, 1 9 6 5 ) . When t h e a c t i v i t i e s o f PDase a nd a c i d p h o s p h a t a s e w e r e m e a s u r e d u n d e r c o n d i t i o n s w h e r e t h e a c c e s s o f s u b s t r a t e t o enzyme was u n r e s t r i c t e d b y a l i m i t i n g membrane, a 2 . 5 - 3 - f o l d i n c r e a s e i n PDase a nd a 2 - f o l d i n c r e a s e i n a c i d p h o s p h a t a s e o v e r t h e i r " f r e e " l e v e l s was o b s e r v e d i n m i t o c h o n d r i a l - l y s o s o m a l f r a c t i o n s f r o m t h e g u i n e a p i g ( T a b l e V I I I ) . The d e g r e e o f a c t i v a t i o n o f t h e s e enzymes was n o t a s g r e a t i n t h e r a t p a r t i c u l a t e f r a c t i o n s n o r was t h e a d v a n t a g e i n a c t i v a t i o n d i s p l a y e d b y f r a c t i o n I V o v e r t h e o t h e r f r a c t i o n s a s g r e a t a s i t was i n t h e c a s e o f t h e g u i n e a p i g . T i " ; " ' c a n bs s e e E f f e c t o f T r i t o n WR-1339 on t h e e q u i l i b r i u m d e n s i t y o f p a r t i c u l a t e - PDase I I P r o b a b l y t h e m o s t c o n c l u s i v e e v i d e n c e r e g a r d i n g t h e T a b l e V I I I . The d e g r e e o f a c t i v a t i o n o f PDase I T and a c i d p h o s p h a t a s e b y T r i t o n X-100 i n p a r t i c u l a t e f r a c t i o n s o f i n t e s t i n a l e p i t h e l i a l c e l l s . The d e g r e e o f a c t i v a t i o n was o b t a i n e d b y d i v i d i n g t h e t o t a l a c t i v i t y o f a f r a c t i o n by i t s " f r e e " a c t i v i t y . The l a t t e r was d e t e r m i n e d i n t h e p r e s e n c e o f 0.3M s u c r o s e w i t h o u t added d e t e r g e n t . The means (±S.D . I f o r t h e numbers o f e x p e r i m e n t s shown i n p a r e n t h e s e s a r e g i v e n . D e g r e e o f A c t i v a t i o n G u i n e a P i g " R a t F r a c t i o n PDase C51 A c i d P h o s . C41 PDase (5). A c i d P h o s . (.41 I 1.9/7 + 0.88 1.73 + 0.32 1.Q1 + Q.83 0.96 + 0.16 I I 2.09 + 0.15 1.33 + 0.15 1.30. + 0.47 1.13 + 0.29 I I I 2.50 + 0.21 2.00 + 0.29. 1.57 + 0.39 1.41 + 0.31 I V 3.05 + 0.58 2.06 + 0.14 1.94 + 0.59 1.21 + 0.23 V 1.64 + 0.28 1.36 + 0.17 1.81 + 0.87 1.03 + 0. 09 66 a s s o c i a t i o n o f a component w i t h l y s o s o m e s i n v o l v e s u s e o f t h e n o n i o n i c d e t e r g e n t T r i t o n WR-1339. T h i s compound, when i n j e c t e d i n t o a n i m a l s , i s t a k e n up by l y s o s o m e s t h e r e b y c a u s i n g a s e l e c t i v e d e c r e a s e i n t h e i r e q u i l i b r i u m d e n s i t y i n s u c r o s e g r a d i e n t s ( W a t t i a u x e t a l . , 1 9 6 3 ) . I f an enzyme i s a s s o c i a t e d w i t h t h e s e g r a n u l e s i t s p o s i t i o n i n t h e g r a d i e n t w i l l s h i f t a c c o r d i n g l y . T h i s c a n be s e e n f r o m t h e r e s u l t s o f a c o n t r o l e x p e r i m e n t p e r f o r m e d w i t h l i v e r ( F i g . 6) w h i c h , as shown by t h e r e s u l t s o f L e i g h t o n e t aJL. (1968) has p r o v e d t o be a u s e f u l t i s s u e f o r t h i s t y p e o f work. The r e s u l t s f r o m an u n i n j e c t e d a n i m a l show t h a t b o t h s u c c i n a t e d e h y d r o g e n a s e and PDase I I a c t i v i t i e s s e d i m e n t c l o s e t o t h e b o t t o m o f t h e c e n t r i f u g e t u b e w h e r e a s t h e d a t a o b t a i n e d w i t h t h e T r i t o n - i n j e c t e d a n i m a l i n d i c a t e t h e r e s u l t w h i c h w o u l d be e x p e c t e d f r o m t h e e x p e r i m e n t s o f v a n Dyck & W a t t i a u x (1968) and E r e c i n s k a e t a l . (1969); t h e p o s i t i o n o f s u c c i n a t e d e h y d r o g e n a s e was u n c h a n g e d whereas t h a t o f PDase I I was s h i f t e d t o a l o w e r d e n s i t y . A c i d p h o s p h a t a s e a l s o was d i s p l a c e d t o a s i m i l a r e x t e n t ( n o t shown). The r e s u l t s o f an e x p e r i m e n t w i t h a g u i n e a p i g i n t e s t i n e a r e d e p i c t e d i n F i g . 7. I n a g r a d i e n t p r e p a r e d w i t h m a t e r i a l f r o m a c o n t r o l a n i m a l s u c c i n a t e d e h y d r o g e n a s e a c t i v i t y banded a t a d e n s i t y o f 1.178 w h i l e t h e a c i d p h o s p h a t a s e and PDase a c t i v i t i e s e x h i b i t e d b r o a d peak.s 67 i i i i i i 0 50 100 0 50 100 distance from top of gradient ('/) F i g . 6: S u c r o s e ^ d e n s i t y - g r a d i e n t c e n t r i f u g a t i o n o f " m i t o c h o n d r i a l -l y s o s o m a l " f r a c t i o n s f r o m g u i n e a p i g l i v e r . G u i n e a p i g l i v e r h o m o g e n a t e s w e r e p r e p a r e d a n d f r a c t i o n a t e d a s d e s -c r i b e d b y deDuve e t a l . (1955) and c o m b i n e d " m i t o c h o n d r i a l -l y s o s o m a l " f r a c t i o n s w e r e a p p l i e d t o 2 0 % - 5 0 % l i n e a r s u c r o s e g r a d i e n t s as d e s c r i b e d i n t h e M a t e r i a l s ^ a n d M e t h o d s s e c -t i o n . The d a t a o n t h e r i g h t (B) w e r e o b t a i n e d f r o m an a n i m a l w h i c h r e c e i v e d a s i n g l e i n t r a p e r i t o n e a l i n j e c t i o n o f T r i t o n WR-1339 (1.3 mg/g b o d y w e i g h t ) d i s s o l v e d i n 0.9% N a C l , 4 d a y s p r i o r t o s a c r i f i c e , w h i l e t h e i n f o r m a -t i o n o n t h e l e f t (A) came f r o m a c o n t r o l a n i m a l t h a t r e c e i v e d a s i m i l a r s i z e d i n j e c t i o n o f 0.9% N a C l a l o n e . R e l a t i v e c o n c e n t r a t i o n v a l u e s w e r e c a l c u l a t e d by d i v i d i n g t h e enzyme c o n c e n t r a t i o n i n a p a r t i c u l a r f r a c t i o n (C) b y t h e i n i t i a l c o n c e n t r a t i o n (C^) i t w o u l d h a v e h a d i f i t h a d b e e n d i s t r i b u t e d h o m o g e n e o u s l y t h r o u g h o u t t h e w h o l e g r a d i e n t . The d i s t a n c e s f r o m t h e t o p o f t h e g r a d i e n t ( a b s c i s s a ) w e r e e x p r e s s e d a s a p e r c e n t a g e o f t h e v a l u e o f t h e w h o l e g r a d i e n t : no c o r r e c t i o n was made f o r t h e s p h e r i c i t y o f t h e t u b e b o t -tom. The r e c o v e r i e s o f s u c c i n a t e d e h y d r o g e n a s e a c t i v i t y i n A and B w e r e 108% and 86% r e s p e c t i v e l y ; t h o s e o f PDase I I w e r e 9 4 % and 1 0 1 % . V e r y s i m i l a r r e s u l t s h a v e b e e n o b -t a i n e d w i t h r a t l i v e r p r e p a r a t i o n s . 68 A B - i 1 i r 1.24 r ' ' 1 I I I 0 50 100 0 50 100 distance from top of gradient (I) F i g . 7: S u c r o s e d e n s i t y - g r a d i e n t c e n t r i f u g a t i o n o f f r a c t i o n s I I I + I V f r o m g u i n e a p i g i n t e s t i n a l e p i t h e l i a l c e l l homogen-a t e s . P o r t i o n s o f t h e p a r t i c u l a t e f r a c t i o n w e r e a p p l i e d t o 2 0 % - 5 0 % s u c r o s e g r a d i e n t s a s d e s c r i b e d i n t h e M a t e r i a l s a n d M e t h o d s s e c t i o n . The r e c o v e r i e s o f SDH i n A and B w e r e 9 1 % a n d 84% r e s p e c t i v e l y ; t h o s e o f a c i d p h o s . w e r e 106% a n d 117% a n d t h o s e o f PDase I I w e r e 89% a n d 94 % r e s p e c t i v e l y . O t h e r e x p e r i m e n t a l d e t a i l s a r e g i v e n i n t h e l e g e n d t o F i g . 6. 69 w i t h m e d i a n d e n s i t i e s c l o s e t o 1.20. W i t h a s i m i l a r f r a c t i o n p r e p a r e d f r o m a g u i n e a p i g i n j e c t e d w i t h T r i t o n WR-1339, t h e s u c c i n a t e d e h y d r o g e n a s e a c t i v i t y was a g a i n f o u n d a t a d e n s i t y c l o s e t o 1.18 w h e r e a s t h e a c i d p h o s p h a t a s e and PDase I I a c t i v i t i e s b a n d e d a t d e n s i t i e s o f 1.168 a nd 1.152 r e s p e c t i v e l y . T h e s e two e x p e r i m e n t s p e r f o r m e d u n d e r s i m i l a r c o n d i t i o n s w i t h l i v e r and i n t e s t i n e p r e p a r a t i o n s o f f e r an i n t e r e s t i n g c o m p a r i s o n o f t h e s e d i m e n t a t i o n b e h a v i o u r o f m i t o c h o n d r i a a n d l y s o s o m e s f r o m t h e two t i s s u e s . L i v e r m i t o c h o n d r i a e x h i b i t a h i g h e r medium d e n s i t y t h a n do t h o s e 3 3 f r o m i n t e s t i n e (1.225g/cm c o m p a r e d t o 1.180g/cm ) , w h e r e a s t h e l y s o s o m e s f r o m b o t h o r g a n s ( c o n t r o l v a l u e s ) a p p e a r t o be r a t h e r s i m i l a r ( F i g s . 6 , 7 ) . The d i f f e r e n c e i n t h e e x t e n t o f t h e l y s o s o m e d e n s i t y s h i f t i n l i v e r a n d i n t e s t i n e upon i n j e c t i o n o f T r i t o n WR-1339 c o u l d be due t o a number o f f a c t o r s e . g . (a) d i f f e r e n c e s i n t h e number and (b) t y p e o f l y s o s o m e s (de Duve & W a t t i a u x , 1966; K o e n i g , 1969) i n b o t h o r g a n s o r (c) d i f f e r e n c e s i n t h e r e l a t i v e u p t a k e o f t h e d e t e r g e n t . I n an e f f o r t t o o b t a i n a b e t t e r a p p a r e n t s h i f t o f i n t e s t i n a l l y s o s o m e s a s e c o n d e x p e r i m e n t was p e r f o r m e d u s i n g a s h a l l o w e r d e n s i t y g r a d i e n t ( F i g . 8,H The b i m o . d a l J n a t u r e o f t h e a c i d p h o s p h a t a s e a n d PDase I I p a t t e r n s i n t h e c o n t r o l e x p e r i m e n t ( F i g . 8A) was a p p a r e n t l y c a u s e d b y t h e l a r g e s t e p i n d e n s i t y a t t h e t o p o f t h e g r a d i e n t . The 70 A B i 1 1 i 1 1 1.24 r i - 1 1 i ; i i 0 5 0 100 0 50 100 distance from top of gradient (7.) F i g . 8: F r a c t i o n a t i o n o f f r a c t i o n s I I I + I V f r o m g u i n e a p i g i n t e s -t i n a l e p i t h e l i a l c e l l h o m o g e n a t e s o n a s h a l l o w s u c r o s e d e n s i t y - g r a d i e n t . The e x p e r i m e n t was i d e n t i c a l t o t h a t d e s c r i b e d i n F i g . 7 e x c e p t t h a t a 3 0 % - 5 0 % l i n e a r s u c r o s e g r a d i e n t was u s e d a nd 20 f r a c t i o n s f r o m e a c h g r a d i e n t w e r e c o l l e c t e d a f t e r w a r d s . S a m p l e s o f t h e m a t e r i a l c o l -l e c t i n g a t t h e " s t e p " i n e a c h d e n s i t y g r a d i e n t ( f r a c t i o n 3) w e r e p r o c e s s e d f o r e l e c t r o n m i c r o s c o p y a s d e s c r i b e d i n t h e M e t h o d s s e c t i o n a n d t h o s e r e s u l t s a r e i l l u s t r a t e d i n F i g s . 9 a nd 10. 71 m a t e r i a l c o l l e c t i n g h e r e c o n s i s t e d m o s t l y o f s m a l l l y s o s o m e s a nd v e s i c l e s a s s e e n b y e l e c t r o n m i c r o s c o p y ( F i g . 9). R e l a t i v e c o n c e n t r a t i o n v a l u e s o f b o t h a c i d p h o s p h a t a s e and PDase I I i n t h e " s t e p " m a t e r i a l f r o m an i n j e c t e d a n i m a l , h o w e v e r , w e r e a b o u t t h r e e t i m e s t h o s e o f t h e c o n t r o l v a l u e s . M o r e o v e r a n a l y s i s o f t h i s m a t e r i a l b y e l e c t r o n m i c r o s c o p y i n d i c a t e d t h e p r e s e n c e o f l a r g e v a c u o l a r o r g a n e l l e s ( F i g . 10) w h i c h w e r e v e r y s i m i l a r t o t h e " T r i t o n - f i l l e d l y s o s o m e s " d e s c r i b e d b y L e i g h t o n e t a l . (1968) . The s h i f t o f g u i n e a p i g i n t e s t i n a l P Dase I I a c t i v i t y o b s e r v e d i n t h e s e e x p e r i m e n t s t o a l o w e r d e n s i t y r e g i o n o f t h e s u c r o s e g r a d i e n t p r o v i d e s s t r o n g e v i d e n c e o f a l y s o s o m a l l o c a t i o n . I n s i m i l a r e x p e r i m e n t s p e r f o r m e d w i t h f r a c t i o n s f r o m r a t i n t e s t i n e no s h i f t i n e i t h e r PDase I I o r a c i d p h o s p h a t a s e c o u l d be d e t e c t e d . Hence f i r m c o n c l u s i o n s a b o u t t h e l o c a t i o n o f t h e r a t enzyme w e r e n o t p o s s i b l e . S o m e ; ; p r o p e r t i e s o f g u i n e a p i g a n d r a t P D a s e I I I n v i e w o f t h e d i f f e r e n c e s n o t e d i n t h e s u b c e l l u l a r d i s t r i b u t i o n o f t h e s e e n z y m e s , some b i o c h e m i c a l p r o p e r t i e s o f e a c h w e r e c o m p a r e d . 72 F i g . 9: E l e c t r o n microscopy of s u b c e l l u l a r p a r t i c l e s from the i n t e s t i n a l e p i t h e l i a l c e l l o f an u n i n j e c t e d guinea p i g . The m a t e r i a l was from f r a c t i o n 3 of the g r a d i e n t i l l u s -t r a t e d i n F i g . 8A (X47,500). 73 10: E l e c t r o n m i c r o s c o p y o f s u b c e l l u l a r p a r t i c l e s f r o m t h e i n t e s t i n a l e p i t h e l i a l c e l l s o f a g u i n e a p i g w h i c h had b e e n i n j e c t e d w i t h T r i t o n WR-1339. The m a t e r i a l was f rom f r a c t i o n 3 o f t h e g r a d i e n t i l l u s t r a t e d i n F i g . 8B (X47,500) . 74 I n f l u e n c e o f pH • The e f f e c t o f pH o n t h e h y d r o l y s i s o f t h y m i d i n e 3 1 - ( 2 , 4 - d i n i t r o p h e n y l ) p h o s p h a t e b y e x t r a c t s o f f r a c t i o n I I I f r o m g u i n e a p i g and r a t i s shown i n F i g . 1 1 . I n t h e a b s e n c e o f SO 2. - , t h e r e was c o n s i d e r a b l e h y d r o l y s i s o f t h e s u b s t r a t e a t l o w pH v a l u e s , e s p e c i a l l y i n t h e c a s e o f t h e g u i n e a p i g . T h i s was p r o b a b l y due t o DNAase I I i n t h e s e p r e p a r a t i o n s s i n c e i t was n o t s e e n i n t h e p r e s e n c e o f SO2. , an i n h i b i t o r o f DNAase I I ( R a u e n b u s c h & A l t m a n , 1 9 6 0 ) . I n a d d i t i o n , a few p r e l i m i n a r y e x p e r i m e n t s seemed t o i n d i c a t e t h a t DNAase I I a c t i v i t y was more e x t e n s i v e a t pH v a l u e s 2.5-4 i n g u i n e a p i g p r e p a r a t i o n s t h a n i n f r a c t i o n s p r e p a r e d f r o m r a t s t h u s a c c o u n t i n g f o r t h e d i f f e r e n c e b e t w e e n t h e a n i m a l s s e e n a t l o w pH v a l u e s i n F i g . 11A. The PDase a c t i v i t i e s o f g u i n e a p i g and r a t p r e -p a r a t i o n s e x h i b i t e d v e r y s i m i l a r p r o f i l e s b u t t h e maximum a c t i v i t i e s w e r e f o u n d a t pH 6.2-6.3 f o r t h e r a t and pH 6.6-6.8 f o r t h e g u i n e a p i g ( F i g s . 11A and B ) . T h i s s m a l l d i f f e r e n c e was a l s o o b s e r v e d when o t h e r f r a c t i o n s f r o m b o t h a n i m a l s w e r e t e s t e d . D e s p i t e t h e s e d i f f e r e n c e s i t was p o s s i b l e t o a s s a y t h e maximum a c t i v i t y o f b o t h enzymes a t pH 6.1 i n s o d i u m s u c c i n a t e b u f f e r , a s d e s c r i b e d i n t h e M a t e r i a l a n d M e t h o d s s e c t i o n , b e c a u s e g u i n e a p i g i n t e s t i n a l P D a s e I I e x h i b i t e d a r e l a t i v e l y f l a t a c t i v i t y p r o f i l e a t pH v a l u e s b e t w e e n 6.0 a n d 6.6 i n t h a t b u f f e r . 75 pH F i g . 1 1 : I n f l u e n c e o f pH o n t h e a c t i v i t y o f r a t a n d g u i n e a p i g P D a s e I I . U s i n g M e t h o d I I t h e e n z y m i c h y d r o l y s i s o f dTpDNP (0.4 3mM) was m e a s u r e d i n m i x t u r e s c omposed o f 25mM s o d i u m s u c c i n a t e - 2 5 m M s o d i u m p h o s p h a t e - 2 5 m M T r i s -HC1 b u f f e r a d j u s t e d t o d i f f e r e n t pH v a l u e s . The a s s a y s w e r e done a t 25°C i n t h e a b s e n c e (A) o r p r e s e n c e (B) o f 125mM (NH 4) SO^ a n d t h e pH o f e a c h r e a c t i o n m i x t u r e was m e a s u r e d p r i o r t o s t o p p i n g t h e r e a c t i o n . A s o n i c a t e d e x t r a c t o f f r a c t i o n I I f r o m e a c h a n i m a l was u s e d . B, r a t enzyme ( 5 8 y g o f p r o t e i n ) ; ©, g u i n e a p i g enzyme (8 9 y g o f p r o t e i n ) . 76 The s t a b i l i t y o f t h e r a t a c t i v i t y i n s o l u t i o n s o f d i f f e r e n t pH i s shown i n F i g , . H'2. An a p p a r e n t a c t i v a t i o n , o b s e r v e d a t pH v a l u e s b e t w e e n 4 a n d 5, c o u l d be due t o t h e d e s t r u c t i o n o f a n i n h i b i t o r o f t h e enzyme ( p e r h a p s a p r o t e a s e ) . Some e v i d e n c e was o b t a i n e d t h a t t h e g u i n e a p i g enzyme was a l s o i i m o r e s t a b l e a t a c i d i c pH v a l u e s ( s e e b e l o w ) . H e a t s t a b i l i t y The P D a s e a c t i v i t i e s o f g u i n e a p i g a n d r a t p r e -p a r a t i o n s d i f f e r e d m a r k e d l y i n t h e i r t h e r m a l s e n s i t i v i t i e s ( F i g . 1 3 ) . H e a t i n g a t 60°C f o r 1 2 m i n r e d u c e d g u i n e a p i g a c t i v i t y t o 2 0 - 28% o f i t s o r i g i n a l v a l u e , w h i l e o n l y 0-2% o f t h e a c t i v i t y o f r a t p r e p a r a t i o n s r e m a i n e d a f t e r h e a t i n g f o r h a l f t h a t t i m e u n d e r t h e s e c o n d i t i o n s . B e c a u s e t h e g u i n e a " p i g a c t i v i t y e x h i b i t e d a r e c o g n i z a b l e "PDase" p r o f i l e e v e n a f t e r h e a t i n g f o r 2 0min a t 60°C an d pH 7 ( F i g . 14) i t i s n o t l i k e l y t h a t t h e i n d i c a t e d s t a b i l i t y was due t o a c o n t a m i n a t i n g enzyme. F u r t h e r i n v e s t i g a t i o n showed t h a t t h e d e s t r u c t i o n o f t h e g u i n e a p i g a c t i v i t y was d e p e n d e n t on b o t h t h e c o m p o s i t i o n and pH o f t h e b u f f e r ; a s s e e n i n F i g . 15 t h e enzyme was more s t a b l e i n T r i s - H C l b u f f e r (pH 7) c o m p a r e d t o p h o s p h a t e b u f f e r (pH 7) a n d more s t a b l e s t i l l a t pH 4. H o w e v e r , h e a t i n g a t t h e l o w e r pH a p p e a r s t o c a u s e a c o n -77 F i g . 12: The s t a b i l i t y o f r a t i n t e s t i n a l PDase I I a c t i v i t y a t v a r i o u s pH v a l u e s . P o r t i o n s of a s o l u b l e enzyme pre -p a r a t i o n ( f r a c t i o n VI) were in c u b a t e d f o r 24h a t 4 C i n b u f f e r mixtures composed of 25mM sodium succinate-25mM sodium phosphate-25mM T r i s - H C l a t d i f f e r e n t pH v a l u e s . Samples of the s o l u t i o n s were then assayed f o r PDase I I a c t i v i t y and compared with the value o b t a i n e d from a c o n t r o l sample, prepared i n water. The pH of the con-t r o l was 7 and i t s PDase I I a c t i v i t y d i d not vary over the i n c u b a t i o n p e r i o d . 7 8 0 4 8 12 min F i g . 13: The s t a b i l i t y of r a t and guinea pig PDase II a c t i v i t y at 60°C. A sonicated extract of f r a c t i o n III was used i n each case. The preparations i n 10 mM sodium phosphate buffer pH 6 . 5 were heated at 60°C and samples were re-moved at the indicated times, cooled rapidly i n ice and assayed at 37°C using Method I. •, rateenzyme ( 5 8 y g of protein); •, guinea pig enzyme ( 8 9 u g of protein). 79 0 L 1 1 1 — : 1 I 4 5 6 7 8 ? pH F i g . 14: I n f l u e n c e of pH on the a c t i v i t y of h e a t - t r e a t e d guinea p i g PDase I I . The experiment was c a r r i e d out u s i n g a s o n i c a t e d f r a c t i o n I I I e x t r a c t . • , unheated samples; r , samples heated f o r 8 min. a t 60°C; A , samples heated f o r 20 min. a t 60°C. The s o l i d curves r e p r e s e n t data o b t a i n e d i n 10 mM sodium phosphate b u f f e r pH 6.5 whereas the hatched curve was obtained i n an experiment where the p r e p a r a t i o n was a c i d i f i e d to pH 4 w i t h a c e t i c a c i d p r i o r t o h e a t i n g . Other d e t a i l s are g i v e n i n the legends t o F i g s . 12 and 13. 80 F i g . 15: V a r i a t i o n ^ i n the s t a b i l i t y o f guinea p i g PDase I I a c t i v -i t y a t 60 C. The experiment was c a r r i e d out as d e s c r i b e d i n the legend to F i g . 13 except t h a t the b u f f e r s i n which the guinea p i g p r e p a r a t i o n were A, lOmM sodium phos-phate pH 7; B, lOmM T r i s - H C l pH 7 and C, lOmM sodium phos-phate a c i d i f i e d to pH 4 wit h a c e t i c a c i d . 81 s i d e r a b l e change i n the enzyme s i n c e the p H - a c t i v i t y curve of a p r e p a r a t i o n heated at 60°C and pH 4 was q u i t e d i f f e r e n t from the normal one ( F i g . 14). The DNAase peak was unchanged but the PDase optimum was s h i f t e d t o pH 8. However, i n t e r p r e t a t i o n of these r e s u l t s was d i f f i c u l t because the i n d i c a t e d s t a b i l i t y at pH 4 ( F i g . 15) was not r e a l l y apparent i n the second experiment ( F i g . 14). M o l e c u l a r s i z e T h i s p r o p e r t y of the two PDases was f i r s t i n v e s t -i g a t e d by sucrose d e n s i t y - g r a d i e n t c e n t r i f u g a t i o n ( F i g . 16). When a p o r t i o n o o f f r a c t i o n VI from both animals was c e n t r i f u g e d on 5-20% sucrose g r a d i e n t , the PDase I I a c t i v i t y e s p e c i a l l y i n the case o f the guinea p i g was spread throughout the g r a d i e n t ( F i g . 16A, B ) . However upon c a r r y i n g out the experiment at a r e l a t i v e l y h i g h i o n i c s t r e n g t h a very d i f f e r e n t r e s u l t was o b t a i n e d , both PDase a c t i v i t i e s were now found a t a p o s i t i o n c l o s e t o the top of the c e n t r i f u g e tube ( F i g . 16C & D) . The small d i f f e r e n c e i n sedimentation r a t e t h a t was observed between the enzymes was r e p r o d u c i b l e ( F i g . 16C & D). From t h i s experiment i t was apparent t h a t i n low i o n i c s t r e n g t h b u f f e r s both of these enzymes e x i s t i n aggregated forms. Gel f i l t r a t i o n chromatography was a l s o used t o i n v e s t i g a t e the m o l e c u l a r weights of the enzymes. These experiments were c a r r i e d out at h i g h i o n i c s t r e n g t h 82 i 1 r f r a c t i o n n o . F i g . 16: Sucro s e d e n s i t y - g r a d i e n t c e n t r i f u g a t i o n o f f r a c t i o n VI from r a t and g u i n e a p i g e p i t h e l i a l c e l l homogenates. I n A and B t h e 5%-20% s u c r o s e g r a d i e n t was p r e p a r e d i n 10 mM sodium phosphate pH 6.5 whereas i n C and D t h e s o l v e n t was 10 mM sodium phosphate - 1 M NaCl pH 6.5. The r e c o v e r i e s o f PDase I I i n A, B, C and D were 71%, 65%, 114% and 86% r e s p e c t i v e l y . •, r a t PDase I I ; •, g u i n e a p i g PDase I I . 83 because of the apparent aggregation phenomenon noted above. The e l u t i o n p o s i t i o n s of the r a t and guinea p i g enzymes from a c a l i b r a t e d Sephadex G-150 column c o r r e s -ponded t o molecular weights of 170,000 and 125,000 r e s p e c t i v e l y ( F i g . 17). T h i s was a s u r p r i s i n g r e s u l t because i n the sedimentation experiments j u s t d e s c r i b e d the guinea p i g enzyme sedimented at a s l i g h t l y f a s t e r r a t e than t h a t from the r a t i n d i c a t i n g t h a t i t probably was a s l i g h t l y l a r g e r molecule. However, i t i s p o s s i b l e t h a t the anomalies i n the s i z e d e t e r m i n a t i o n s of the enzymes were due t o s u b t l e d i f f e r e n c e s i n the shapes of the molecules. D i s c u s s i o n The experiments o f van Dyck & Wattiaux (1968) and E r e c i n s k a e t a l . (1969) w i t h r a t l i v e r p r o v i d e strong evidence t h a t PDase I I i s lysosomal. Others, however, have r e p o r t e d the presence o f the enzyme i n other c e l l f r a c t i o n s i n a v a r i e t y of t i s s u e s ; f o r example, i n the m i t o c h o n d r i a . o f hog kidney ( R a z z e l l , 1961); i n the s o l u b l e f r a c t i o n o f r a t pancreas homogenates (van V e n r o o i j & Poort, 197 0) and i n the n u c l e a r f r a c t i o n of salmon t e s t i s p r e -p a r a t i o n s (Menon & Smith,1970). In the p r e s e n t case a broad d i s t r i b u t i o n of the enzyme i n s u b c e l l u l a r f r a c t i o n s of i n t e s t i n a l e p i t h e l i a l c e l l s was a l s o found (Flanagan & 84 1.0 1.5 2.0 v e /v 0 F i g . 17: M o l e c u l a r weight d e t e r m i n a t i o n of i n t e s t i n a l PDase I I from the r a t , and guinea p i g by g e l f i l t r a t i o n . The e l u t i o n volumes (V ) of the p r o t e i n s were determined on a Sephadex G-150 column (2.5cm X 35cm) w i t h 20mM sodium a c e t a t e - l M NaCl p H 5 as the e l u a n t as d e s c r i b e d i n the Methods s e c t i o n . Blue Dextran 2000 was used to e s t i -mate the excluded volume (V ) and s o n i c a t e d e x t r a c t s of f r a c t i o n I I I were the sources of PDase I I . A , a l d o l a s e (mol. wt. 158000); •, E. c o l i a l k a l i n e phos-phatase (mol. v/t. 80000); o , ovalbumin (mol. wt. 45000); A , chymotrypsinogen (mol. wt. 25000); B , r a t PDase I I ; ©, guinea p i g PDase I I . 85 Zbarsky, 1973; 1974). However, the highest s p e c i f i c a c t i v i t y of the enzyme was always found in those fractions which were enriched with lysosomes. In addition, the guinea pig enzyme followed a s h i f t i n lysosomal density when the animals were injected with the nonionic detergent T r i t o n WR-1339, a finding considered conclusive evidence of a lysosomal location (see Beaufay, 1972). However, the locationoof the rat enzyme i s less obvious from our r e s u l t s . While the s p e c i f i c a c t i v i t y was again high i n the "mitochondrial-lysosomal" f r a c t i o n , a large portion (40%) of the t o t a l a c t i v i t y appeared i n the soluble f r a c t i o n of the homogenate. This d i s t r i b u t i o n was similar to that previously reported for rat mucosal scrapings (Flanagan, 1970; Hynie & Zbarsky, 1970a) although less soluble a c t i v i t y was found i n the present case. A difference between the latency of guinea pig and rat acid hydrolases was also noted, i n agreement with the r e s u l t s of other investigators. For instance, Hubscher et aJL. (1965) showed that treatment of subcellular fractions from guinea pig inte s t i n e with a Waring Blendor produced a modest increase i n the a c t i v i t i e s of a number of acid hydrolases while lack of a latency e f f e c t has been reported for these enzymes i n i n t e s t i n a l preparations from the rabbit (Porteous & Clark, 1965) and the rat (Gresham & Pover, 1967). In t h i s connection i t may be s i g n i f i c a n t that we were unable to demonstrate, i n the r a t 86 the s h i f t i n d e n s i t y o f p a r t i c u l a t e a c i d phosphatase and PDase I I upon T r i t o n WR-1339 a d m i n i s t r a t i o n which we observed w i t h the guinea p i g . Since lysosomes have been r e p e a t e d l y seen i n the e p i t h e l i a l c e l l s of r a t i n t e s t i n e (Behnke & Moe, 1964; Moe e t a l . , 1965; T r i e r , 1968), i t i s p u z z l i n g t h a t a c i d h y d r o l a s e s from these c e l l s d i d not d i s p l a y the behaviour expected f o r lysosomal enzymes. P o s s i b l y t h i s i n d i c a t e s t h a t the gr a n u l e s i n t h i s t i s s u e were more e a s i l y damaged by the ma n i p u l a t i o n s used to prepare the t i s s u e f r a c t i o n s than those from guinea p i g e p i t h e l i u m , s i n c e i t i s r e c o g n i z e d t h a t lysosomes from d i f f e r e n t sources v a r y g r e a t l y i n t h e i r f r a g i l i t y ( T u r n b u l l & N e i l , 1969; Beaufay, 1972; Reid, 1972). Hsu and Tappel (1964) have r e p o r t e d t h a t i n t e s t i n a l e p i t h e l i u m , i n common w i t h kidney (Straus, 1956), c o n t a i n s a more heterogeneous p o p u l a t i o n o f lysosomes than does l i v e r . We have observed t h a t the p a r t i c u l a t e d i s t r i b u t i o n o f a c i d phosphatase and PDase i n i n t e s t i n a l e p i t h e l i u m were not i d e n t i c a l > g r a n u l e s c o n t a i n i n g PDase I I appeared to sediment s l i g h t l y f a s t e r than those c o n t a i n i n g a c i d phosphatase. Hubscher e t al_. (1965) have a l s o noted t h a t some p a r t i c l e s c o n t a i n i n g a c i d h y d r o l a s e s sedimented from mucosal homogenates at low c e n t r i f u g a l f o r c e s . Perhaps the s i t u a t i o n i n the i n t e s t i n e i s somewhat analogous to t h a t i n the l i v e r where a c i d phosphatase and a c i d DNAase are con t a i n e d i n d i f f e r e n t p o p u l a t i o n s of lysosomes^ 87 (Beaufay e t a l . , 1964) . F i n a l l y , as de Duve e t 'al. (1955) have p o i n t e d out the " f r e e " a c t i v i t y of enzymes can become r e d i s t r i b u t e d amongst the f r a c t i o n s as a r e s u l t of a d s o r p t i o n and other phenomena d u r i n g f r a c t i o n a t i o n s t u d i e s . However, such a r t i f a c t s were probably not important i n the pr e s e n t study s i n c e i n experiments to t e s t the p o s s i b i l i t y of a d s o r p t i o n o n l y f r a c t i o n I adsorbed any PDase I I a c t i v i t y from a s o l u b l e f r a c t i o n o f e p i t h e l i a l c e l l s (Table I X ) . The PDases of guinea p i g and r a t i n t e s t i n e d i f f e r e d i n o ther p r o p e r t i e s a l s o . The r a t enzyme had a higher m o l e c u l a r weight as i n d i c a t e d by g e l f i l t r a t i o n , was more l a b i l e at 60°C and e x h i b i t e d a lower pH optimum. One e x p l a n a t i o n f o r these observed d i f f e r e n c e s i s t h a t the a c t i v i t i e s s t u d i e d a r i s e from two d i f f e r e n t p r o t e i n s . However oth e r p o s s i b i l i t i e s which should a l s o be c o n s i d e r e d are t h a t the d i f f e r e n c e s were due to the presence of contaminating substances i n the r e l a t i v e l y crude p r e p a r a t i o n s used i n these experiments, or to d i f f e r e n c e s i n the p h y s i c a l s t a t e o f the enzymes, s i n c e a g r e a t e r tendency of the guinea p i g PDase I I t o aggregate was observed. PART BY PURIFICATION OF INTESTINAL PDASE T I Experiments u s i n g r a t p r e p a r a t i o n s I n i t i a l attempts were made to p u r i f y the r a t enzyme because s o l u b l e preparations: c o u l d be e a s i l y o b t a i n e d from 88 Table IX. T e s t for a d s o r p t i o n of s o l u b l e PDase TT by p a r t i c u l a t e f r a c t i o n s . F r a c t i o n s I-V were prepared as d e s c r i b e d under " S u b c e l l u l a r f r a c t i o n a t i o n " and each was then resugpended i n 5ml p o r t i o n s o f f r a c t i o n VI. A f t e r l h a t 0 C the suspensions were c e n t r i f u g e d a t 1-05, OOOg f o r 60min and the PDase IT a c t i v i t y was estimated T n the supernatants and compared to the* amount of s o l u b l e PDase I I a c t i v i t y o r i g i n a l l y p r e s e n t . F r a c t i o n Percentage of o r i g i n a l PDase IT a c t i v i t y I 73 I I 107 I I I 120 IV 153 V 100 89 t h i s s o u r c e ( P a r t A ) . However b e f o r e a r e l i a b l e p u r -i f i c a t i o n p r o c e d u r e was worked o u t a number o f p r e l i m i n a r y e x p e r i m e n t s y i e l d e d some i n t e r e s t i n g f i n d i n g s . P r e c i p i t a t i o n by ammonium s u l p h a t e When p o r t i o n s o f a h i g h - s p e e d s u p e r n a t a n t were m i x e d w i t h a p p r o p r i a t e amounts o f s o l i d ( N H i f ) 2 S O i t and t h e p r e c i p i t a t e d and s o l u b l e p r o t e i n s s e p a r a t e d by c e n t -r i f u g a t i o n t h e r e s u l t s d e p i c t e d i n F i g . 18 were o b t a i n e d . I t a p p e a r e d t h a t t h e b u l k o f t h e PDase I I a c t i v i t y c o u l d be p r e c i p i t a t e d between 35% and 55% s a t u r a t i o n w i t h ( N H i t ) 2 S O i t . When t h e e x p e r i m e n t was c a r r i e d o u t i n a more c o n v e n t i o n a l manner by a d d i n g amounts o f (NHit) 2 S O 4 t o a s i n g l e p o r t i o n o f h i g h - s p e e d s u p e r n a t a n t and r e m o v i n g t h e p r e c i p i t a t e d p r o t e i n s a s t h e y were s a l t e d o u t , t h e r e s u l t s were n o t r e p r o d u c i b l e and t h e y i e l d s o f p r e c i p i t a b l e enzyme a c t i v i t y were q u i t e v a r i a b l e . The d a t a f r o m two s u c h e x p e r i m e n t s a r e shown i n T a b l e X. W h i l e t h e r e s u l t s o f e x p e r i m e n t 1 ( T a b l e X) r e s e m b l e t h o s e i n F i g . 18, t h e r e was an u n a c c o u n t a b l e l o s s o f a c t i v i t y i n e x p e r i m e n t 2. F o r t h e s e r e a s o n s p r e c i p i t a t i o n by ammonium s u l p h a t e was n o t a d o p t e d a s a r o u t i n e p r o c e d u r e i n t h e p u r i f i c a t i o n o f PDase I I . I n some o f t h e s e e x p e r i m e n t s i t was a l s o f o u n d t h a t m ost o f t h e p o r t i o n o f PDase I I a c t i v i t y n o t p r e c i p i t a t e d by 55-60% (NHit). 2 S C K r e m a i n e d s o l u b l e u n t i l 100% s a t u r a t i o n 90 120 i "O (D i _ CD > O O CD C 13 O E CO 60 T— i — i — i — i — r ~ T PDase 0 10 20 25 30 35 40 45 50 55 60 65 70 75 10 20 25 30 35 40 45 50 55 60 65 70 75 amm. sulph. sat.-^ F i g . 1 8: Ammonium s u l p h a t e f r a c t i o n a t i o n o f t h e s u p e r n a t a n t f r a c t i o n o b t a i n e d f r o m a r a t i n t e s t i n a l h o m o g e n a t e . A p p r o p r i a t e amounts o f (WA^) . w e r e a d d e d t o p o r t i o n s o f a s u p e r -n a t a n t f r a c t i o n w h i c h e a c h c o n t a i n e d 6 u n i t s o f PDase I I a c t i v i t y . The h i s t o g r a m s d i s p l a y t h e amounts o f PDase I I and p r o t e i n r e c o v e r e d i n t h e s u p e r n a t a n t f r a c t i o n ( c l e a r ) a n d p r e c i p i t a t e ( s h a d e d ) o f e a c h s t e p . O t h e r d e t a i l s a r e g i v e n i n t h e M a t e r i a l s a n d M e t h o d s s e c t i o n . 91 Table X. Ammonium s u l p h a t e p r e c i p i t a t i o n o f r a t i n t e s t i n a l PDase,II. F r a c t i o n VI was prepared as d e s c r i b e d i n the M a t e r i a l s and Methods s e c t i o n . One r a t was used i n Expt. 1 w h i l e 3 r a t s were used i n Expt. 2. PDase IT a c t i v i t y (units) (NH^^SO^ P r e c i p i t a t e d S o l u b l e f r a c t i o n (%sat.) Expt. 1 Expt. 2 Expt. 1 Expt. 2 0 - - 103.4 368.9 0 — 35 4.9 40.0 104.7 340.1 35 - 55 66.0 80.0 46.3 82.8 55 - 80 21.3 - 24.4 -92 was reached. I t was thought t h a t t h i s might i n d i c a t e the presence of more than one PDase a c t i v i t y i n the p r e p a r a t i o n . T h i s p o s s i b i l i t y was i n v e s t i g a t e d by the method of zone p r e c i p -i t a t i o n (Porath, 1962) which has a l s o been c a l l e d s o l u b i l i t y chromatography by Hoffman & McGivern ( 1 9 6 9 ) . Zone p r e c i p i t a t i o n T h i s procedure i s based on the f a c t t h a t the p r o t e i n s are e l u t e d f a s t e r than s a l t s from g e l f i l t r a t i o n columns. I f a s o l u t i o n o f s a l t which i s a l s o a p r o t e i n p r e c i p i t a t i n g agent (e.g. ammonium sulphate) i s a p p l i e d to a column of Sephadex f o l l o w e d by a s o l u t i o n c o n t a i n i n g a mixture o f p r o t e i n s , the p r o t e i n s . w i l l r a p i d l y c a t c h up" with the zone of p r e c i p i t a t i n g agent ( F i g . 1 9 ) . Thus the e l u t i o n o f the v a r i o u s p r o t e i n components from the column w i l l be r e t a r d e d t o the exte n t t h a t they are s a l t e d out by the p r e c i p i t a t i n g agent. A g r e a t e r s e p a r a t i o n of the components i s obtained by u s i n g a g r a d i e n t o f the p r e c i p i t a n t (Porath,1962; Hoffman & McGivern, 1 9 6 8 ) . Using t h i s technique i t should be p o s s i b l e t o f i n d out i f PDase I I a c t i v i t y i s i n f a c t p r e c i p i t a t e d a t d i f f e r e n t ammonium sulphate s a t u r a t i o n s as suggested by the f i n d i n g s shown i n F i g . 18 and Table X. The r e s u l t s of two zone p r e c i p i t a t i o n experiments are i l l u s t r a t e d i n Fig.s 20A & B l In the f i r s t ( F i g . 2QA1 a g r a d i e n t o f 93 Concentres fion A + B + C ^ Precipitating agent Distance from fop of the column a) C one en fra fion Precipitating agent Distance from fop of the column b) F i g . 19: The p r i n c i p l e of zone p r e c i p i t a t i o n . The schematic drawing was taken from Porath (1962). Three substances A, B and C have d i f f e r e n t s o l u b i l i t i e s i n the p r e c i p i t a t i n g agent, a) s t a r t i n g c o n d i t i o n s ; b) i n the f i r s t phase of washing the substances, A, B and C a l l move f a s t e r than the grad-i e n t substance. A f t e r r e a c h i n g the g r a d i e n t they d i s p l a y d i f f e r e n t behaviour; C moves independently of the g r a d i e n t , A and B are r e t a r d e d t o d i f f e r e n t e x t e n t s . 94 fraction no. F i g . 20: Zone p r e c i p i t a t i o n of r a t i n t e s t i n a l PDase I I . The exper-iment was c a r r i e d out as d e s c r i b e d i n the M a t e r i a l s and Methods s e c t i o n u s i n g p o r t i o n s of a f r a c t i o n VI prepared from a r a t homogenate. In A the d e c r e a s i n g (NH4)2SO4 g r a d i e n t was from 3 M to 0 M whereas i n B i t went from 1.5 M to 0 M. The A280 p r o f i l e i s shown by the h i s t o -grams; •, (NH 4) 2S04; PDase I I . 95 3M-*-0M (NHij.) 2 S O V was a p p l i e d t o t h e G-100 column w h e r e a s i n t h e s e c o n d ( F i g . 20B) a s h a l l o w e r g r a d i e n t o f 1.5M>0M was u s e d . N e i t h e r e x p e r i m e n t g a v e any i n d i c a t i o n o f t h e r e b e i n g any more t h a n one PDase component i n t h e s e i n t e s t i n a l p r e p a r a t i o n s . The p o s s i b i l i t y o f u s i n g zone p r e c i p i t a t i o n t o p u r i f y PDase I I was r a i s e d by t h e s e e x p e r i m e n t s b e c a u s e u n d e r some c o n d i t i o n s ( e . g . F i g . 2OB) a good s e p a r a t i o n o f PDase I I f r o m o t h e r i n t e s t i n a L . p r o t e i n s c o u l d be o b t a i n e d . A few p u r i f i c a t i o n experiments',were c a r r i e d o u t w i t h m o d e r a t e s u c c e s s b u t t h e method was j u d g e d t o o cumbersome and w a s t e f u l o f ammonium s u l p h a t e f o r r o u t i n e p u r i f i c a t i o n o f t h e enzyme. A d s o r p t i o n on A g a r o s e D u r i n g e x p e r i m e n t s t o d e t e r m i n e t h e m o l e c u l a r w e i g h t o f t h e r a t enzyme by g e l f i l t r a t i o n (see P a r t A) a sample w h i c h had been c o n c e n t r a t e d by ammonium s u l p h a t e p r e c i p i t a t i o n was a p p l i e d t o a 10% a g a r o s e column ( B i o - G e l A-0.5M) and e l u t e d w i t h 50mM sodium a c e t a t e pH 5. The a c t i v i t y emerged f r o m t h e column v e r y c l o s e t o t h e i n c l u d e d volume (V^) i n d i c a t i n g an a p p a r e n t low m o l e c u l a r w e i g h t f o r t h e enzyme ( F i g . 2 1 A ) . T h i s was u n e x p e c t e d s i n c e a m o l e c u l a r w e i g h t o f 160,000-170,000 had b e e n o b t a i n e d on Sephadex G-150 colu m n s ( F i g . 171. Xt was 96 0 CO CC i _ CD -4—' co CD T*3 O JZ CL CO O JO CL 50mM N a O A c pH5 B / IN 50mM N a O A c - 0 . 5 M NaCl pH5 D —\ o f—«-Q 13 0 V „ / V 0.5 1.0 't F i g . 21 C h r o m a t o g r a p h y o f r a t i n t e s t i n a l P Dase I I on a g a r o s e . The p r o t e i n s w h i c h p r e c i p t a t e d b e t w e e n 3 5 % a n d 55% s a t u r a t i o n w i t h (NH^^SO^ f r o m a h i g h - s p e e d s u p e r -n a t a n t f r a c t i o n w e r e r e d i s s o l v e d i n e i t h e r 50mM s o d i u m a c e t a t e pH 5 o r i n 50mM s o d i u m a c e t a t e - 0 . 5 M N a C l pH 5. The r e s u l t s i n A a n d C w e r e o b t a i n e d when p r o t e i n s o f t h e s e s o l u t i o n s w e r e s e p a r a t e l y a p p l i e d t o a c o l u m n (2.5cm X 35cm) o f B i o - G e l A-0.5m u s i n g t h e e l u a n t s i n d i c a t e d . The l i g h t l y h a t c h e d c u r v e r e p r e s e n t s t h e e l u t i o n p r o f i l e o f (NH^^SO^ a s d e t e r m i n e d by c o n d u c -t i v i t y m e a s u r e m e n t s . I n B and D t h e p r o t e i n s a m p l e s w e r e d i a l y s e d a g a i n s t t h e e l u t i n g b u f f e r b e f o r e a p p l i c a t i o n t o t h e c o l u m n . 97 thought t h a t perhaps the ammonium sulphate had caused some k i n d of d i s s o c i a t i o n of the enzyme and so another p o r t i o n of the same p r e p a r a t i o n was d i a l y z e d f r e e of ammonium sulphate and r e a p p l i e d t o the same agarose column. S u r p r i s i n g l y almost none of the PDase I I a c t i v i t y was e l u t e d from the column ( F i g . 21B). However the phospho-d i e s t e r a s e a c t i v i t y c o u l d be recovered by e l u t i n g the column w i t h a b u f f e r of h i g h i o n i c s t r e n g t h . Furthermore i t was found t h a t i f a b u f f e r of high i o n i c s t r e n g t h was used to e l u t e the agarose columns, the enzyme was/elttifefijd i n a p o s i t i o n t o i n d i c a t e a m o l e c u l a r weight of about 150,000 and the r e c o v e r y of PDase a c t i v i t y from the columns was between 90 and 100% even i f the a p p l i e d p r e p a r a t i o n s c o n t a i n e d c o n s i d e r a b l e amounts of ammonium sulphate ( F i g . 21C & D). A p p a r e n t l y the phosphodiesterase i n these p r e -p a r a t i o n s was adsorbed to the agarose by f o r c e s which were e l i m i n a t e d or minimised in ::the presence of h i g h c o n c e n t r a t i o n s of s a l t . T h e r e f o r e when the f r a c t i o n which contained ammonium sulphate was chromatographed ( F i g . 21A), the PDase was adsorbed on the column u n t i l the zone of (NHif)2S0i+ s t a r t e d t o e l u t e whereupon i t became r a p i d l y desorbed i n an anomo.lous e l u t i n g p o s i t i o n . T h i s a d s o r p t i o n of PDase I I on B i o - G e l agarose columns which was a l s o seen w i t h Pharmacia agarose p r e p a r -a t i o n s (Sepharose) was used w i t h g r e a t e f f e c t i n p u r i f y i n g the enzyme and w i l l be r e f e r r e d t o l a t e r . 98 Ion exchange chromatography Salmon t e s t e s PDase II.has been r e a d i l y p u r i f i e d by chromatography on CMC a t pH 6.3 (Menon & Smith, 1970) whereas B e r n a r d i & B e r n a r d i (1966) showed t h a t the enzyme from hog spleen was r e t a i n e d on a DEAE-Sephadex column at pH 6.8. T h e r e f o r e i t was a s u r p r i s e t o f i n d , i n p r e -l i m i n a r y experiments, t h a t i n t e s t i n a l PDase I I as w e l l as the bulk of the p r o t e i n i n these p r e p a r a t i o n s was not adsorbed a t pH 6.3 to e i t h e r CMC or DEAEC. L a t e r i t was found t h a t the enzyme was s t r o n g l y bound to CMC o n l y a t pH v a l u e s l e s s than 5.5. However the low degree of a d s o r p t i o n to DEAEC a t pH 6.3 was not c o r r e c t e d by i n c r e a s i n g the pH a t which the chromatography was c a r r i e d out but was the r e s u l t of another phenomenon. I t has been found i n our l a b o r a t o r y t h a t most r a t i n t e s t i n a l p r o t e i n s g e n e r a l l y are not bound to the f i b r o u s forms of DEAEC (e.g. Whatman DE22 and DE23) whereas they are r e a d i l y adsorbed t o the m i c r o g r a n u l a r forms (DE32 and DE52) of t h i s anion exchanger (Krasny, J . , Poulson, R., F r i z e l l , J . and Flanagan, P. unpublished o b s e r v a t i o n s ) . T h e r e f o r e when s o l u b l e p r e p a r a t i o n s from r a t s were chromatographed on Whatman DE32 FDase I I was c o n s i s t e n t l y adsorbed s t r o n g l y a t pH v a l u e s h i g h e r than 6.5. Thus d e s p i t e the e a r l i e r n e g a t i v e r e s u l t i s o b t a i n e d a t pH 6.3 i t was p o s s i b l e to develop a scheme f o r the p u r i f i c a t i o n of r a t i n t e s t i n a l 99 PDase I I which i n v o l v e d a d s o r p t i o n of the a c t i v i t y on both DEAEC and CMC. P u r i f i c a t i o n of the r a t enzyme The procedure used was a combination of the methods d e s c r i b e d i n the p r e v i o u s s e c t i o n and i n v o l v e d chromatography on DEAEC, CMC and agarose columns. A t y p i c a l experiment i s d e p i c t e d i n F i g . 22. The s t a r t i n g m a t e r i a l was a supernatant f r a c t i o n o b t ained by u l t r a -c e n t r i f u g a t i o n of a homogenate of r a t i n t e s t i n a l mucosa. In the e a r l y experiments t h i s f r a c t i o n corresponded to f r a c t i o n VI of Table vlfBuit many of the l a t e r experiments were c a r r i e d out w i t h a more e a s i l y prepared e x t r a c t as d e s c r i b e d i n the M a t e r i a l s and Methods s e c t i o n . In most of the experiments the i n i t i a l p u r i f i c a t i o n step (chromatography on DEAEC) was c a r r i e d out u s i n g a "batch" technique, but o c c a s i o n a l l y the p r o t e i n s o l u t i o n was a p p l i e d t o the DEAEC column d i r e c t l y . A f t e r e l u t i o n of the column w i t h a l i n e a r g r a d i e n t of sodium phosphate, the f r a c t i o n s c o n t a i n i n g PDase a c t i v i t y were combined and the pH o f the s o l u t i o n a d j u s t e d t o 5 w i t h g l a c i a l a c e t i c a c i d . O c c a s i o n a l l y , i f the p r o t e i n content was v e r y high, a f a i n t opalescence developed at t h i s stage and t h i s was removed by c e n t r i f u g a t i o n . The a c i d i f i e d f r a c t i o n was then a p p l i e d to a CMC column. Most of the p r o t e i n s were not 100 . DEAEC I \ 20mM Sod. Phosph pH6-5 1-6 0-8 0 E c O 00 C N z < co oc o CO CO < 0-4r-0-2 CMC 50mM Sod. Acet pH 50 LL i X -I 0-4 -J.0-2 u c 1-6 W 0-2 20 40 60 80 FRACTIONS-20ml BIO OR A-0-5m 50mM Sod. Acet. 1 80mM Sod. Chlor. 1 pH 50 1 ,....n --• . i 1V 0-8 -JO 13-2 2-4 0-2 0-1 i 20 40 60 80 FRACTIONS-10ml 100 —'0 co < on LU I— co LLJ Q O I o_ CO O X a. 1-6 0-8 F i g . 22: Chromatographic p u r i f i c a t i o n of r a t i n t e s t i n a l PDase I I . The p r e p a r a t i o n o f the e x t r a c t (5 r a t s were used) and the chromatographic steps are d e s c r i b e d i n the M a t e r i a l s and Methods s e c t i o n . The f r a c t i o n s from DEAEC chromatography c o n t a i n i n g PDase I I a c t i v i t y were combined and a c i d i f i e d w i t h a c e t i c a c i d to pH 5. T h i s s o l u t i o n was then a p p l i e d t o the CMC column and the a c t i v e f r a c t i o n s e l u t e d t h e r e -from were combined and d i l u t e d w i t h d i s t i l l e d water u n t i l the c o n d u c t i v i t y of the p r o t e i n s o l u t i o n was equal to t h a t of the e l u t i n g b u f f e r shown f o r the B i o - G e l step; the s o l u -t i o n was then a p p l i e d to t h i s column. The r e c o v e r i e s o f p r o t e i n and enzyme a c t i v i t y a t the v a r i o u s steps are g i v e n i n Table XI (expt. 1 ) . 101 bound to the column and appeared i n the flow-through volume w h i l e the PDase I I a c t i v i t y was adsorbed s t r o n g l y and c o u l d be e l u t e d :>;with 0. 2M NaCl. The f i n a l s tep o f the p u r i f i c a t i o n u t i l i z e d the agarose a d s o r p t i o n phenomenon d e s c r i b e d p r e v i o u s l y . The a c t i v e f r a c t i o n s from the CMC column were combined and d i l u t e d w i t h c o l d (4°C) d i s t i l l e d water u n t i l the [NaCl] was approximately 0.05M. A l t e r n a t i v e l y , the combined f r a c t i o n was d i a l y z e d a g a i n s t the e l u t i n g b u f f e r f o r the agarose chromatography. Only very small amounts of p r o t e i n were adsorbed to the agarose column whereas 70-95% of the PDase I I was bound at these s a l t c o n c e n t r a t i o n s . The enzyme a c t i v i t y was e l u t e d w i t h a g r a d i e n t o f [NaCl] o r , more g e n e r a l l y , by i n c r e a s i n g the [NaCl] of the e l u t i o n b u f f e r t o 0.5M i n a s i n g l e s t e p . The r e s u l t s of a number of these p u r i f i c a t i o n experiments are summarized i n Table XI. Phosphodiesterase I I was p u r i f i e d 350-550-fold by t h i s procedure i n y i e l d s which v a r i e d between 20-40%. Perhaps because the f i n a l p r e p a r a t i o n s c ontained such s m a l l amounts of p r o t e i n f u r t h e r i n c r e a s e s i n s p e c i f i c a c t i v i t y were not measurable u s i n g c o n v e n t i o n a l chromatographic techniques such as chromato-graphy on h y d r o x y a p a t i t e , p h o s p h o c e l l u l o s e or re-chromato-graphy on DEAEC. However, chromatography on DNA-cellulose gave an i n t e r e s t i n g r e s u l t . 102 T a b l e XX.. P u r i f i c a t i o n of r a t i n t e s t i n a l PPase IT Tne experiments were c a r r i e d out as o u t l i n e d i n the t e x t u s i n g the chromatographic procedures d e s c r i b e d i n the M a t e r i a l s and Methods s e c t i o n . The column chromato-graphy f o r Expt. 1 i s shown i n F i g . 22s. Over- F o l d T o t a l T o t a l S p e c i f i c a l l r e - p u r i f -F r a c t i o n -&cA©;t^. (units) p r o t e i n a c t i v i t y covery i c a t n . (mg) (units/mg) (%) supernatant 710 920 0.77 100 1 Expt.1 DEAEC 500 117 4.3 70 6 C5rats) CMC 434 20.8 21 61 27 agarose 305 0.7 420 43 .',540 supernatant 1150 1613 0.71 100 1 Expt.2 DEAEC 725 360 2. 0. 63 3 C5rats) CMC 598 60.2 10 52 14 agarose 493 2.4 207 43 290 supernatant 1800 1930 0.93 100 1 Expt. 3 DEAEC 615 225 2.7 34 3 (IQrats) CMC 502 156 3.2 28 3 .5 agarose 351 1.0 338 20 360 supernatant 1680 6540 0.65 100 1 Expt. 4 DEAEC 879 283 3.1 52 5 (20rats) CMC 574 336.0 16 34 25 agarose 546 1.5 355 33 550 103 D N A - c e l l u l o s e chromatography I t was c o n s i d e r e d improbable t h a t i n t e s t i n a l PDase I I would b i n d t o DNA-cellulose because the enzyme, being an exonuclease, would be expected to have a low a f f i n i t y f o r DNA. However when the experiments were performed ( F i g . 23; Table XII) i t was found t h a t a v a r i a b l e p o r t i o n of h i g h l y p u r i f i e d PDase was s t r o n g l y adsorbed t o these columns. The r e s u l t s i n F i g . 23 show moreover t h a t the unadsorbed p o r t i o n o f PDase was not due t o o v e r l o a d i n g of these s m a l l columns s i n c e when r e a p p l i e d on a f r e s h column i t again appeared i n the flow-through volume. Since the f r a c t i o n o f the PDase adsorbed to DNA-cellulose d i d not c o n t a i n d e t e c t a b l e p r o t e i n (Table XII) i t s s p e c i f i c a c t i v i t y was ve r y h i g h . In a d d i t i o n , the s i z e of t h i s adsorbed f r a c t i o n seemed to vary c o n s i d e r a b l y with the age o f the p r e p a r a t i o n used because f r e s h p r e p a r a t i o n s c o n t a i n e d a g r e a t e r p o r t i o n o f PDase capable o f being bound t o DNA - c e l l u l o s e (Table X I I I ) . C h a r a c t e r i z a t i o n o f the adsorbed and non-adsorbed peaks of PDase w i l l be d e s c r i b e d l a t e r . Experimentsi'musing guinea p i g p r e p a r a t i o n s E x t r a c t i o n of,,guinea p i g i n t e s t i n a l PDase I I Because some d i f f i c u l t y had been encountered d u r i n g some of the experiments d e s c r i b e d i n P a r t A i n o b t a i n i n g 104 F i g . 23: D N A - c e l l u l o s e c h r o m a t o g r a p h y o f p u r i f i e d r a t PDase I I . The e x p e r i m e n t shown i n A was c a r r i e d o u t a s d e s c r i b e d i n t h e M e t h o d s s e c t i o n w i t h 233 u n i t s o f p u r i f i e d PDase I I . The e l u t i n g b u f f e r was 5 mM T r i s - 5 mM NaH^PO^ - 10 mM N a C l pH 7.6 and 2 m l f r a c t i o n s w e r e c o l l e c t e d . The numbers e n c l o s e d i n t h e f i g u r e s r e f e r t o t h e m i l l i m o l a r N a C l c o n -y c e n t r a t i o n s o f t h e b u f f e r s u s e d t o e l u t e t h e c o l u m e i n a s t e p w i s e manner. The f i r s t p e a k o f PDase I I t o b e ~ e l u t e d ( n o n - a d s o r b e d f r a c t i o n ) was r e a p p l i e d t o a s e c o n d DNA-c e l l u l o s e c o l u m n i n t h e e x p e r i m e n t i l l u s t r a t e d i n B. O t h e r d e t a i l s a r e g i v e n i n T a b l e X I I . 105 Table tKTT. D N A - c e l l u l o s e chromatography of p u r i f i e d r a t PDase IT. The experiment was c a r r i e d out as d e s c r i b e d i n the M a t e r i a l s and Methods s e c t i o n -using a f r e s h p r e p a r a t i o n o f PDase I I . PDase I I S p e c i f i c F r a c t i o n ^ " Y ? * ? p r o t e i n a c t i v i t y , ( u n i t s ] (mgl. (units/mg) a p p l i e d 233 0. .60. 39 0 unadsorbed 107 0. ,25 420 • adsorbed 117 <0. .0.5 > 23 0.0. Table X I I I . Comparison of the behaviour, o f f r e s h and "aged" p r e p a r a t i o n s of p u r i f i e d r a t PDase TT on D N A - c e l l u l o s e . F r e s h p r e p a r a t i o n s were not more than 2 weeks o l d . "Aging" of p u r i f i e d PDase IT was c a r r i e d out as-d e s c r i b e d i n the M a t e r i a l s and Methods s e c t i o n . PDase a c t i v i t y C%1 P r e p a r a t i o n Unadsorbed Adsorbed F r e s h F r e s h "Aged" 56 48 9-0 43 52 10 106 t r u l y s o l u b l e e x t r a c t s o f guinea p i g PDase I I , a v a r i e t y of methods was i n i t i a l l y attempted t o extract, the enzyme from p a r t i c u l a t e f r a c t i o n s of homogenates of guinea p i g mucosa. A summary of the r e s u l t s i s g i v e n i n Table XIV. None of the treatments was completely e f f e c t i v e i n e x t r a c t i n g the PDase from guinea p i g s u b c e l l u l a r p a r t i c l e s . I t i s l i k e l y however t h a t combination of a number of the e x t r a c t i o n procedures might have improved the e f f i c i e n c y of e x t r a c t i o n o f PDase but t h i s was not attempted because of the p o s s i b i l i t y of i n t e r f e r e n c e i n the l a t e r chromato-g r a p h i c s t e p s . For example when 4M urea e x t r a c t s o f the gr a n u l e s were chromatographed on DEAEC columns the PDase a c t i v i t y was not adsorbed. E x t r a c t s o f the guinea p i g p a r t i c u l a t e f r a c t i o n s were prepared i n s t e a d by repeated s o n i c a t i o n i n T r i s - H C l b u f f e r a t pH 8 as d e s c r i b e d i n the M a t e r i a l and Methods s e c t i o n . The f i n a l sediment remaining a f t e r c a r r y i n g out the e x t r a c t i o n procedure con-t a i n e d l e s s than 3 0% o f the o r i g i n a l PDase I I a c t i v i t y (Table XIV). However i t should be mentioned t h a t i f these " s o l u b i l i z e d " guinea p i g f r a c t i o n s were kept f o r a few days a t 4°C up to 4 0% of t h e i r a c t i v i t y c o u l d be sedimented by u l t r a c e n t r i f u g a t i o n . T h e r e f o r e i t i s n o t l l i k e l y t h a t these p r e p a r a t i o n s were t r u l y s o l u b l e but r a t h e r t h a t they c o n s i s t e d o f v e r y : s m a l l d i s r u p t e d fragments caused by the s o n i c a t i o n procedure. 107 T a b l e XIV. S o l u b i l i z a t i o n o f guinea p i g PDase I T The p a r t i c u l a t e p r e p a r a t i o n was- obtained by combining f r a c t i o n s IT + ITT + IV as d e s c r i b e d i n the Methods s e c t i o n . The p a r t i c l e s were resuspended i n 5ml of e i t h e r 20mM sodium a c e t a t e pH 5 or 20mM T r i s - H C l pH 8 c o n t a i n i n g the a p p r o p r i a t e a d d i t i o n and allowed t o stand a t 24°C f o r 3 0min. T r y p s i n , neuraminidase and phospholipase C i n c u b a t i o n s were c a r r i e d out as d e s c r i b e d by Henning e t a l . (1973). A 3.5h i n c u b a t i o n o f phospholipase D a t pH 5 and 37°C i n the presence of 0.07M C a C l 2 was used. At the end o f each treatment the p a r t i c u l a t e matter was sedimented by c e n t r i f u g a t i o n and resuspended i n 20mM T r i s - H C l pH 8 (5ml). PDase a c t i v i t y i n the supernatant and sediment was d e t e r -mined u s i n g Method I. Percentage o f — recovered a c t i v i t y Treatment Recovery (%')> Supernatant Sediment 2 0mM T r i s - H c l pH 8 100 21 79 2 0mM sodium a c e t a t e pH 5 100 2 98 3 x s o n i c a t e (pH 8) 90 72 28 50mM mercaptoethanol 89 41 59 50mM EDTA 85 8 92 10% t-amyl a l c o h o l 65 48 52 3M NaCl 56 18 82 1% deoxycholate 49 38 62 t r y p s i n (lOOug/ml) 80 41 59 neuraminidase (5units/ml) 83 11 89 phospholipase C (lOOvig/mll 73 20 80 phospholipase D (lOOug/ml) 66 18 82 4M urea 73 51 4 9 1% T r i t o n X-100 111* 37 * 63 141* '••65* - 35 i n t e r f e r e n c e i n PDase I I assay 108 P u r i f i c a t i o n o f the gu i n e a - p i g enzyme The p u r i f i c a t i o n procedure again u t i l i z e d chromato-graphy on DEAEC, CMC and agarose. I t was necessary to c a r r y out the DEAEC step a t pH v a l u e s h i g h e r than 7 because the bulk of the p r o t e i n s i n the guinea p i g e x t r a c t were i n s o l u b l e below t h i s v a l u e . A s o n i c a t e d e x t r a c t o f f r a c t i o n s I I + I I I + IV from the guinea p i g were c a r r i e d through the p u r i f i c a t i o n procedure and f o r comparison a r a t e x t r a c t c o n t a i n i n g s i m i l a r amounts of p r o t e i n was p u r i f i e d on an i d e n t i c a l s et of columns. The r e s u l t s are shown i n F i g s . 24, 25 and 26 and summarized i n Table XV. In the DEAEC and CMC p u r i f i c a t i o n steps the behaviour o f the guinea p i g enzyme was reasonably s i m i l a r t o t h a t o f the r a t enzyme although l e s s guinea p i g PDase a c t i v i t y was recovered from these columns (Table XV). A l a r g e d i f f e r -ence o c c u r r e d at the agarose s t e p . Whereas the r a t enzyme was almost e n t i r e l y adsorbed o n l y 10% o f the guinea p i g PDase remained on the agarose, the balance appearing i n the flow-through volume wi t h the bulk of the p r o t e i n ( F i g . 26). T h i s d i f f e r e n c e was mostly r e s p o n s i b l e f o r the poorer y i e l d and lower degree:, of p u r i f i c a t i o n of the guinea p i g enzyme (Table XV). I t was noted a l s o t h a t i n the steps performed a t pK 5 (CMC and agarose) the guinea p i g p r e p a r a t i o n s were q u i t e o p a l e s c e n t i n d i c a t i n g t h a t not a l l the p r o t e i n o f the s o l u t i o n was completely s o l u b l e . I t was p o s s i b l e to 109 80 120 fraction no. 160 200 o 9 i 1 1 1 1 r— B 1 1 r M o * 1 AJ ' 1 1 10 1 ' ' ' ' ' 0.5 • \ 0 i 1 1—;— i i j i • i 80 120 fraction no. 160 o a a. F i g . 24: DEAEC c h r o m a t o g r a p h y o f r a t a n d g u i n e a p i g i n t e s t i n a l e x t r a c t s . The e x t r a c t s , f r o m 10 r a t s (A) a n d 10 g u i n e a p i g s (B) w e r e p r e p a r e d a n d c h r o m a t o g r a p h e d on DEAEC a t pH 7.6 a s d e s c r i b e d i n t h e M e t h o d s s e c t i o n . F r a c t i o n s o f 20ml w e r e c o l l e c t e d . ©, PDase I I ; O, A 2 8 0 * [ N a C l ] . The r e s u l t s o f t h e e x p e r i m e n t s a r e s e t f o r t h i n T a b l e XV w h e r e t h e r e c o v e r i e s o f P D a s e I I and p r o t e i n a r e g i v e n . 110 F i g . 25: CMC chromatography of p a r t i a l l y p u r i f i e d r a t and guinea p i g PDase I I . The a c t i v e f r a c t i o n s from the DEAEC columns shown i n F i g . 24 were combined, a c i d i f i e d t o pH 5 as des-c r i b e d p r e v i o u s l y (see F i g . 22), c l a r i f i e d by c e n t r i f u -g a t i o n and chromatographed on CMC as d e s c r i b e d i n the M a t e r i a l s s e c t i o n . Twenty m i l l i l i t r e f r a c t i o n s were c o l -l e c t e d . A, r a t ; B, guinea p i g ; •, PDase I I ; o, A 2 8 0 ; —,— [NaCl]. Other d e t a i l s may be found i n Table XV. I l l F i g . 26: Chromatography of p a r t i a l l y p u r i f i e d r a t and guinea p i g PDase II on agarose. The a c t i v e f r a c t i o n s from the CMC columns shown i n F i g . 25 were combined, c o n c e n t r a t e d by u l t r a f i l t r a t i o n , d i a l y s e d a g a i n s t s e v e r a l changes of 50mM sodium acetate-50mM NaCl pH 5 (the e l u t i n g b u f f e r ) and chromatographed on agarose columns as d e s c r i b e d i n the M a t e r i a l s and Methods s e c t i o n . F r a c t i o n s (2 0ml) were c o l l e c t e d , and the adsorbed PDase I I was e l u t e d by i n c r e a s i n g the [NaCl] of the e l u t i n g b u f f e r to 0.5M as i n d i c a t e d . Other d e t a i l s are g i v e n i n Table XV. A, r a t ; B, guinea p i g ; ©, PDase I I ; O, A 0 Q n . 112 Tabl e XV. P u r i f i c a t i o n of PDase from guinea p i g and r a t i n t e s t i n e E x t r a c t s from 10 guinea p i g s and 10 r a t s were prepared as d e s c r i b e d i n the M a t e r i a l s and Methods s e c t i o n . The experiments were performed i n a s i m i l a r f a s h i o n t o those shown i n Ta b l e XT except t h a t DEAEC chromatography was c a r r i e d out a t pH 7.6,as d e s c r i b e d i n the Methods s e c t i o n . The column chromatographic p r o f i l e s o f these experiments are shown i n F i g s . 24, 25 and 26". T o t a l T o t a l S p e c i f i c O v e r a l l F o l d F r a c t i o n a c t i v i t y p r o t e i n a c t i v i t y r e c o v e r y p u r i f i c a t i o n (units) (mg). (units/mg) • C%) Rat Supernatant 1150 1600 0.71 100 1 DEAEC 725 360 2.0 63 2.8 CMC 598 60 9.9 52 14 agarose 493 2.4 2B7.7 43 300 Guinea p i g Supernatant 767 1500 0.51 100 1 DEAEC 308 194 1.6 40 3.1 CMC 232 58 4.0 30 7.8 agarose 20 2.7 7.4 2.6 15 113 c l a r i f y these s o l u t i o n s by u l t r a c e n t r i f u g a t i o n but the r e s u l t i n g supernatant s o l u t i o n s c o ntained o n l y a small amount of the o r i g i n a l PDase I I a c t i v i t y . Since some evidence had a l r e a d y been o b t a i n e d t h a t guinea p i g PDase tends t o aggregate under c e r t a i n c o n d i t i o n s then perhaps t h i s a g g r e g a t i o n / s o l u b i l i t y problem was r e s p o n s i b l e f o r the poorer p u r i f i c a t i o n : d f the guinea p i g enzyme. In any event f u r t h e r attempts to p u r i f y t h i s enzyme were abandoned because of the d i f f i c u l t y i n o b t a i n i n g t r u l y s o l u b l e p r e p a r a t i o n s and because of the low y i e l d s u s i n g c o n v e n t i o n a l p u r i f i c a t i o n procedures. Some p r o p e r t i e s of p a r t i a l l y p u r i f i e d phosphodiesterase I I  from the guinea p i g and r a t Since these enzymes appeared.to be l o c a l i z e d i n m d i f f e r e n t areas of the i n t e s t i n a l e p i t h e l i a l c e l l and»aiso d i s p l a y e d a number of d i s t i n g u i s h a b l e c h a r a c t e r i s t i c s as d e s c r i b e d i n P a r t A, i t was o f i n t e r e s t to i n v e s t i g a t e and compare the p r o p e r t i e s of the p a r t i a l l y p u r i f i e d enzymes. These experiments were c a r r i e d out w i t h prepar-a t i o n s which had been s e q u e n t i a l l y p u r i f i e d on DEAEC, CMC and agarose. pH optimum The p H - a c t i v i t y curves of the p a r t i a l l y p u r i f i e d 114 enzymes ( F i g . 27) were very s i m i l a r t o those d i s p l a y e d by the u n p u r i f i e d enzymes ( F i g . 11). The prominent shoulder of a c t i v i t y n o t i c e a b l e at lower pH valu e s was probably due to contaminating DNAase a c t i v i t y as i l l u s t r a t e d by F i g . 11A). As wit h the crude p r e p a r a t i o n s the optimum pH of the r a t enzyme was s l i g h t l y lower than t h a t of the guinea p i g and w h i l e the l a t t e r d i s p l a y e d about 50% o f i t s maximum a c t i v i t y at pH of about 8.5, the r a t enzyme was v i r t u a l l y i n a c t i v e a t t h i s pH. The optimum pH f o r each of the enzymes was not a p p r e c i a b l y a f f e c t e d by i n c r e a s i n g the c o n c e n t r a t i o n of the b u f f e r s , but the absolute amount of a c t i v i t y found under these c o n d i t i o n s was d i m i n i s h e d c o n s i d e r a b l y (Table XVI).. High c o n c e n t r a t i o n s of sodium s u c c i n a t e b u f f e r i n h i b i t e d both enzyme a c t i v i t i e s , the guinea p i g enzyme to a s l i g h t l y g r e a t e r extent than the r a t enzyme. A s i m i l a r i n h i b i t i o n by h i g h s u c c i n a t e c o n c e n t r a t i o n s has been r e p o r t e d by R a z z e l l and Khorana (1961) f o r s pleen PDase. The e f f e c t s of v a r i o u s compounds on PDase I I a c t i v i t y These compounds were chosen i n the hope t h a t a s e l e c t i v e e f f e c t on one of the enzyme a c t i v i t i e s might be ob t a i n e d thereby d i s t i n g u i s h i n g i t from the ot h e r . The 2 _ r e s u l t s are shown i n Table XVII. As noted i n P a r t A, SCK appears to i n h i b i t DNAase a c t i v i t y without any e f f e c t on 115 i r- i 1 r I I L I 1 1 1 3 4 5 6 7 8 9 pH F i g . 27: I n f l u e n c e of pH on the a c t i v i t y of p u r i f i e d r a t and guinea ppig PDase I I . The experiment was c a r r i e d out as d e s c r i b e d i n the legend t o F i g . 11A wi t h samples of the most p u r i -f i e d r a t (•) and guinea p i g (•) p r e p a r a t i o n s d e s c r i b e d i n Table XV. 116 Table "XVT. The e f f e c t o f s u c c i n a t e c o n c e n t r a t i o n on the a c t i v i t y - o f p u r i f i e d r a t and guinea p i g PDase "IT. The^enzyme p r e p a r a t i o n s were the adsorbed f r a c t i o n s from agarose chromatography. PDase I I was assayed u s i n g Method I except t h a t the b u f f e r c o n c e n t r a t i o n was v a r i e d as i n d i c a t e d . PDase a c t i v i t y (%) [succinate] Rat Guinea p i g 0.1 100 100 0.2 100 103 0.4 92 99. 0.8 84 79 1.0 79 69 117 T a b l e XVII. The e f f e c t s o f urea and v a r i o u s s a l t s on r a t and guinea p i g PDase a c t i v i t y The r a t and guinea p i g p r e p a r a t i o n s were the adsorbed f r a c t i o n s from agarose chromatography. PDase I I was assayed by Method ,1 i n the presence o f the a p p r o p r i a t e a d d i t i o n . The 100% v a l u e s r e p r e s e n t a c t i v i t i e s of Q.16 u n i t s and 0.15 u n i t s o f r a t and guinea p i g PDase IT r e s -p e c t i v e l y . PDase I I a c t i v i t y C%L A d d i t i o n Rat Guinea p i g N o n e 100 100 2M u r e a 84 187 0.1M EDTA 97 90. 0..1M MgCl 2 95 90. 0.02M 10.0 10.0. 0 . . 2 M ( N H i t i a S O i * 97 95 2.0M ( N F U U S O ^ 45 28 6M NaCl 41 32 0.02M N a H z P C K 85 92 0..0.8M N a H a P O i t 81 88 0.16M N a H 2 P O ^ 69 82 0.32M N a H a P C K 64 69. 118 the PDase a c t i v i t y i n the same p r e p a r a t i o n s . O c c a s i o n a l l y however, a small decrease i n PDase was observed i n the presence of 125mM ( N H i t ) 2 S O t t . Therefore i n the experiments d e s c r i b e d here a wide range of sol c o n c e n t r a t i o n s were used. The r e s u l t s i n Table XVII show t h a t s u b s t a n t i a l decreases i n PDase a c t i v i t y were observed i n the presence of h i g h c o n c e n t r a t i o n s of ammonium sul p h a t e . However t h i s i n h i b i t i o n was probably due to the h i g h i o n i c s t r e n g t h of these s o l u t i o n s because sodium c h l o r i d e at a comparable i o n i c s t r e n g t h gave a s i m i l a r degree of i n h i b i t i o n of PDase. I t i s a l s o i n t e r e s t i n g t o note t h a t i n these experiments the guinea p i g enzyme was i n h i b i t e d t o a s l i g h t l y h i g h e r degree by both (NHi* 1 ' ) 2 SO i + and NaCl than was the r a t enzyme. Magnesium ions have been r e p o r t e d by Hilmoe, (1960) t o i n h i b i t PDase I I whereas EDTA i s supposed to a c t i v a t e the enzyme. In the experiments r e p o r t e d here a s m a l l i n h i b i t i o n of both enzymes was observed i n the presence of both compounds (Table XVII). In a d d i t i o n , 2M urea, which was r e p o r t e d by R a z z e l l & Khorana (1961) t o i n h i b i t c a l f s p leen PDase I I by 50% was found to o n l y m i l d l y i n h i b i t the i n t e s t i n a l enzymes from r a t and guinea p i g . Thus taken as a whole the s i m i l a r i t y o f e f f e c t s of a l l these compounds on r a t and guinea p i g PDases i s more s t r i k i n g than are the d i f f e r e n c e s and leads one to b e l i e v e t h a t the enzymes from both animals are q u i t e s i m i l a r . 119 Heat s t a b i l i t y In view of the pronounced t h e r m o s t a b i l i t y d i f f e r -ences which were observed with the crude enzymes e a r l i e r i n t h i s t h e s i s , i t was of i n t e r e s t t o compare t h i s p r o p e r t y of the p a r t i a l l y p u r i f i e d enzymes. I t was i n i t i a l l y found t h a t the p u r i f i e d r a t enzyme was remarkably s t a b l e t o h e a t i n g under c o n d i t i o n s where the crude enzyme was q u i c k l y i n a c t i v a t e d (Table X V I I I ) . The p a r t i a l l y p u r i f i e d p r e -p a r a t i o n was a l s o l e s s s t a b l e t o the h i g h temperatures a t h i g h e r pH v a l u e s (Table X V I I I ) , c o n f i r m i n g r e s u l t s ob-t a i n e d p r e v i o u s l y w i t h crude p r e p a r a t i o n s . When the experiments were performed by h e a t i n g at d i f f e r e n t temperatures f o r a f i v e minute p e r i o d the r e s u l t s shown i n F i g 28 were o b t a i n e d . From these i t can be seen t h a t a f i v e minute p e r i o d a t 64°C was needed t o h a l f -i n a c t i v a t e the p a r t i a l l y p u r i f i e d r a t enzyme whereas the corresponding temperature f o r the guinea p i g p r e p a r a t i o n was 61°C. These v a l u e s can be compared wi t h those obtained w i t h crude f r a c t i o n s i n s i m i l a r experiments; 54°C f o r the r a t ( F i g . 28) and 60°C f o r the guinea p i g . T h e r e f o r e p u r i f i c a t i o n appeared not t o a f f e c t the t h e r m o s t a b i l i t y of the guinea p i g enzyme w h i l e t h a t of the r a t enzyme was s i g n i f i c a n t l y i n c r e a s e d . 120 Table XVI;IT. Comparison of the i n a c t i y a t i o n o f crude and p u r i f i e d r a t PDase I I a t 60 C. The crude supernatant f r a c t i o n was a sample, of f r a c t i o n VI Csee under " S u b c e l l u l a r f r a c t i o n a t i o n " I whereas the p u r i f i e d p r e p a r a t i o n was t h a t f r a c t i o n which was adsorbed d u r i n g agarose chromatography. P o r t i o n s o f each i n lOmM sodium phosphate b u f f e r were heated a t 60 C and a l i q u o t s removed a t t h e times i n d i c a t e d and assayed f o r PDase I I . PDase I I a c t i v i t y remaining C%1 P r e p a r a t i o n 5 min 10. min 20. min Crude supernatant (pH 6.5). 16 4 a P u r i f i e d CpH 6.5) 9.9 95 8.7 P u r i f i e d CpH 7.5) 85 68 58 121 temp.-°C F i g . 2 8: Heat' i n a c t i v a t i o n o f c r u d e a n d p u r i f i e d r a t a n d g u i n e a p i g P D a se I I . P o r t i o n s o f t h e c r u d e ( o p e n s y m b o l s ) a n d a g a r o s e - p u r i f i e d ( c l o s e d s y m b o l s ) p r e p a r a t i o n s d e s c r i b e d i n T a b l e XV w e r e h e a t e d i n 5mM Tris-5mM NaH^PC^ pH 7.6 a t t h e t e m p e r a t u r e s i n d i c a t e d f o r 5min. A f t e r r a p i d c o o l i n g i n i c e - w a t e r , a l i g u o t s w e r e a s s a y e d u s i n g M e t h o d I f o r r e m a i n i n g PDase I I a c t i v i t y . • and B, r a t ; ©, g u i n e a p i g . The r e s u l t s o b t a i n e d w i t h t h e c r u d e g u i n e a p i g p r e p a r a t i o n w e r e i n d i s t i n g u i s h a b l e f r o m t h o s e o b t a i n e d w i t h t h e p u r i f i e d f r a c t i o n (©). 122 E f f e c t of TpDNP c o n c e n t r a t i o n R a z z e l l & Khorana (1961) r e p o r t e d t h a t the M i c h a e l i s constant f o r dTpNP h y d r o l y s i s by p u r i f i e d spleen -3 PDase was 3 x 10 M although Horwitz e t al_. (1972) have -4 p u b l i s h e d a v a l u e of 2£5. x 10 M. F i e r s & Khorana (1963) have shown t h a t the Km f o r h y d r o l y s i s of the same compound by an analogous PDase from L a c t o b a c i l l u s a c i d o p h i l u s was -4 4.3 x 10 M. Although von T i g e r s t r o m & Smith (1969) and Menon & Smith (1970) used dTpDNP as a s u b s t r a t e f o r spleen and t e s t i s PDase I I r e s p e c t i v e l y the a p p r o p r i a t e Km v a l u e s were not g i v e n . The e f f e c t of dTpDNP c o n c e n t r a t i o n on the a c t i v i t y of p u r i f i e d p r e p a r a t i o n s of r a t and guinea p i g PDase I I i s shown i n F i g . 29. The data have j-geen p l o t t e d i n the manner d e s c r i b e d by Lineweaver & Burk (1934) which showed the apparent Km v a l u e s of the ratr . .and guinea -5 -5 p i g enzymes to be 4.1 x 10 M and 2.2 x 10 M r e s p e c t i v e l y . The corresponding V v a l u e s were 57 0 and 6.9 units/mg of * ^ max ^ p r o t e i n r e s p e c t i v e l y . I t i s i n t e r e s t i n g t o note t h a t the Km v a l u e s f o r i n t e s t i n a l PDase I I are one t e n t h to one e i g h t i e t h of those f o r the spleen or L a c t o b a c i l l u s enzymes although some of t h i s d i f f e r e n c e i s probably due to the d i f f e r e n c e i n a f f i n i t y of the enzymes f o r dTpNP and dTpDNP (see under D i s c u s s i o n ) . I t i s not s u r p r i s i n g t h a t the v a l u e s of Vmax were so d i f f e r e n t from eacH other and from those of spleen 123 F i g . 29: E f f e c t of s u b s t r a t e c o n c e n t r a t i o n on p u r i f i e d r a t and guinea p i g PDase I I . The agarose p u r i f i e d r a t (•) and guinea p i g (•) p r e p a r a t i o n s shown i n Tab'le XV were used. PDase I I a c t i v i t y was assayed u s i n g Method I except t h a t the dTpDNP c o n c e n t r a t i o n was v a r i e d as i n d i c a t e d . The s t r a i g h t l i n e s were drawn by the method of l e a s t squares u s i n g a l l the data i n A but onl y the r e s u l t s o b tained a t the s i x lower s u b s t r a t e c o n c e n t r a t i o n s i n B; the c o r r e l a -t i o n c o e f f i c i e n t s were 0.998 i n each case. 124 PDase (3020 units/mg of p r o t e i n ) and L a c t o b a c i l l u s PDase (2 91 units/mg of p r o t e i n ) s i n c e t h i s parameter i s largely-dependent on the degree of p u r i f i c a t i o n of the appro-p r i a t e enzyme p r e p a r a t i o n . The smal l d i f f e r e n c e i n Km noted above w i t h pur-i f i e d r a t and guinea p i g PDases was not r e p r o d u c i b l e w i t h d i f f e r e n t preparations' o f these enzymes. F i g . 30 shows an experiment where the a c t i v i t i e s (units/ml) of the PDases were very s i m i l a r but the s p e c i f i c a c t i v i t i e s o f the p r e p a r a t i o n were, however, q u i t e d i f f e r e n t . From t h i s experiment the apparent Km v a l u e s f o r the r a t and -5 guinea p i g enzymes were found t o be 4.4 x 10 M and 4.9 x 10 ^ r e s p e c t i v e l y . So i t appears t h a t the M i c h a e l i s constant of guinea p i g PDase I I i s i n p a r t dependent on the a c t i v i t y of the p r e p a r a t i o n . In many of these experiments s u b s t r a t e i n h i b i t i o n of enzyme a c t i v i t y c o u l d be d e t e c t e d at dTpDNP c o n c e n t r a t i o n s g r e a t e r than ImM (Figs 29, 30). Sub s t r a t e i n h i b i t i o n of PDase I I has a l s o been observed by F i e r s & Khorana (1963) with the L a c t o b a c i l l u s enzyme where i t a l s o becomes appar-ent over a very narrow c o n c e n t r a t i o n range. E f f e c t o f temperature on the h y d r o l y s i s of dTpDNP When the i n i t i a l v e l o c i t i e s of both PDases were determined a t temperatures from 12°C to 50°C the data -20 0 20 40 60 80 ]A mM"' F i g . 30: E f f e c t o f s u b s t r a t e c o n c e n t r a t i o n o n p a r t i a l l y p u r i f i e d r r a t a n d g u i n e a p i g PDase I I . The enzyme p r e p a r a t i o n s , w h i c h w e r e p a r t i a l l y p u r i f i e d on DEAEC as d e s c r i b e d i n F i g . 24 w e r e d i l u t e d s o t h a t e a c h c o n t a i n e d a p p r o x i m a t e l y 3 u n i t s o f PDase I I / m l . The e x p e r i m e n t was c a r r i e d o u t as d e s c r i b e d i n t h e l e g e n d t o F i g . 29 a n d t h e s t r a i g h t l i n e s w e r e a g a i n d r a w n b y t h e m e t h o d o f l e a s t s q u a r e s u s i n g a l l t h e d a t a e x c e p t t h e r e s u l t o b t a i n e d a t t h e h i g h e s t s u b s t r a t e c o n c e n t r a t i o n ; t h e c o r r e l a t i o n c o e f -f i c i e n t s w e r e 0.994 and 0.995 f o r t h e r a t (•) and g u i n e a p i g (•) p r e p a r a t i o n s r e s p e c t i v e l y . 126 shown i n the A r r h e n i u s p l o t s i n F i g . 31 were o b t a i n e d . An apparent break i n thevejupKe f o r each enzyme was observed. The a c t i v a t i o n energy f o r the PDase-catalyzed h y d r o l y s i s of TpDNP decreased as the r e a c t i o n temperature was in c r e a s e d ; a decrease from 12.8kcal/mol to 7.3kcal/mol was observed f o r the r a t enzyme and i n the case o f the guinea p i g enzyme the Eact decreased from 17.4kcal/mol to 11.9kcal/mol. Temperature "breaks" i n A r r h e n i u s p l o t s are a con-sequence o f a phase change i n e i t h e r the enzyme or the r e a c t a n t s a c c o r d i n g t o Kumamoto et al_. (1971) , assuming t h a t the change i n temperature does not (a) i n a c t i v a t e the enzyme, (b) a l t e r the a f f i n i t y o f the enzyme f o r the s u b s t r a t e , an a c t i v a t o r or an i n h i b i t o r o r (c) a l t e r the pH f u n c t i o n ©Cthe r e a c t i o n components (Raison, 1973). Since i t has a l r e a d y been shown t h a t temperatures above 50°C i n a c t i v a t e the guinea p i g and r a t PDases to v a r y i n g e x t e n t s ( F i g . 2 8) i t might be argued t h a t c o n d i t i o n (a) was not met i n the experiments d e s c r i b e d here. However every e f f o r t was made to ensure t h a t i t was; the i n i t i a l v e l o c i t y a t each temperature was determined immediately a f t e r a d d i t i o n of the enzyme by estimating, the i n i t i a l l i n e a r r e l e a s e of DNP f o r o n l y a very s h o r t time p e r i o d (.generally o n l y 30s to 2min).. A l s o d e s p i t e the f a c t t h a t the V of the r e a c t i o n was not measured at each temperature max 4" as recommended by Dixon & Webb (19641, the same b a s i c 127 °c 60 50 40 30 20 2.4 p 1 1 1 r 06 I l l I I 1 3.0 3.1 3.2 3.3 3.4 3.5 io 3x U F i g . 3 1 : A r r h e n i u s p l o t s o f t h e r a t a n d g u i n e a p i g PDase I I -c a t a l y s e d h y d r o l y s i s r e a c t i o n s . The DEAEC p u r i f i e d r a t (•) a n d g u i n e a p i g (•) p r e p a r a t i o n s shown i n T a b l e XV w e r e e m p l o y e d . E a c h was a s s a y e d u s i n g M e t h o d I a s d e s -c r i b e d i n t h e M a t e r i a l s a n d M e t h o d s s e c t i o n e x c e p t t h a t t h e dTpDNP c o n c e n t r a t i o n was 0.8 mM and t h e t e m p e r a t u r e o f t h e r e a c t i o n was v a r i e d a s i n d i c a t e d . I n e a c h c a s e t t h e r e a c t i o n was i n i t i a t e d b y t h e a d d i t i o n o f t h e enzyme. 128 r e s u l t s were obtained i n .^several experiments performed w i t h TpDNP c o n c e n t r a t i o n s from 0.4mM to l.OmM. That i s to say t h a t . a decrease i n a c t i v a t i o n energy by 30-40% was observed f o r each enzyme as the temperature of the r e a c t i o n was r a i s e d . T h i s break always o c c u r r e d between 30°C and 40°C. D i s c u s s i o n P u r i f i c a t i o n of the enzyme Whereas a number of methods was i n i t i a l l y used i n attempts to p u r i f y these enzymes, namely ammonium sulphate p r e c i p i t a t i o n and zone p r e c i p i t a t i o n , the procedure e v e n t u a l l y adopted employed i o n exchange chromatography on DEAEC and CMC and a novel use of chromatography on agarose (Flanagan & Zbarsky, 197 2). The i n c o n s i s t e n t r e s u l t s o b t ained i n the o r i g i n a l ammonium-.sulphate p r e c i p i t a t i o n experiments were p o s s i b l y due to the i n h i b i t i n g e f f e c t s of h i g h i o n i c s t r e n g t h s o l u t i o n s on the enzymes, although i f t h i s i s so, i t i s p u z z l i n g as to why t h i s e f f e c t was not seen i n e v e r y experiment. High, c o n c e n t r a t i o n s of not o n l y (NHOzSCU but a l s o NaCl and sodium s u c c i n a t e were found to cause con-s i d e r a b l e i n h i b i t i o n o f PDase a c t i v i t y . Chromatography on CMC (Menon & Smith, 1970) or CM-Sephadex (B e r n a r d i & B e r n a r d i , 1968) has been -used to 129 p u r i f y PDase from t e s t i s and spleen r e s p e c t i v e l y . The pH used i n these s t u d i e s was 6.3 a t which v a l u e i n t e s t i n a l PDase I I a p p a r e n t l y i s not p o s i t i v e l y charged s i n c e i t does not bi n d t o CMC. The other commonly used chromatographic technique f o r the p u r i f i c a t i o n of PDases of t h i s type has u t i l i z e d h y d r o x y a p a t i t e (Bernardi & B e r n a r d i , 1966; Menon & Smith, 1970; and van Dyck & Wattiaux, 1970). T h i s was not g e n e r a l l y used i n the present work, however, because the r e s u l t s were not very r e p r o d u c i b l e and low y i e l d s of p u r i f i e d enzyme i n v a r i a b l y were obt a i n e d . The hydroxy-a p a t i t e used i n these experiments (Bio-Gel HTP) was composed of very small c r y s t a l s and i t i s p o s s i b l e t h a t the very slow flow r a t e s of the columns were r e s p o n s i b l e f o r the poor r e s u l t s o b t a i n e d . The use of DEAEC chromatography f o r PDase I I p u r i f i c a t i o n has not been widespread. B e r n a r d i & Ber-n a r d i (1966) r e p o r t e d both spleen RNAase- and spleen PDase I I were adsorbed on DEAE-Sephadex A-50 a t pH 6.8 and c o u l d be e l u t e d s e p a r a t e l y w i t h a 0.01M-0.3M potassium phosphate g r a d i e n t . However s i n c e l a t e r p u b l i c a t i o n s from t h i s group do not mention a step i n v o l v i n g SDEAEC or DEAE-Sephadex one must assume t h a t some d i f f i c u l t y was encoun-t e r e d w i t h the procedure and indeed Menon & Smith (197 0) have shown t h a t t e s t i s PDase was not adsorbed to DEAEC at pH 7.4. In the prese n t work r a t i n t e s t i n a l PDase TT: 130 was adsorbed to DEAEC a t pH v a l u e s o f 6.5 and h i g h e r . At pH 7.5 b e t t e r r e s u l t s were obtained u s i n g T r i s -phosphate b u f f e r r a t h e r than T r i s - H C l b u f f e r , so phosphate appears t o p l a y a s u b t l e b e n e f i c i a l r o l e i n the a d s o r p t i o n -d e s o r p t i o n p r o c e s s . PriGrbably of g r e a t e s t i n t e r e s t i n the c u r r e n t scheme was the step i n v o l v i n g a d s o r p t i o n o f PDase I I a c t i v i t y on agarose columns. T h i s phenomenon, d i s c o v e r e d by a c c i d e n t , made r o u t i n e l y p o s s i b l e a 10-20 f o l d p u r i f i c a t i o n o f the r a t enzyme i n a s i n g l e s t e p . I t i s probable t h a t the a d s o r p t i o n o f the PDase I I a c t i v i t y to the agarose i n v o l v e s i o n i c i n t e r a c t i o n s s i n c e even the p u r e s t p r e p a r a t i o n s of agarose probably s t i l l ; c o n t a i n small numbers of sulphate groups (Hjerten, 1962). In at l e a s t one other case the presence of charged groups on a g e l has proved b e n e f i c i a l . B e t t e r s e p a r a t i o n o f h i s t o n e s has been obtained w i t h o l d r a t h e r than new batches of B i o - G e l P-10 (S. McLeod, p e r s o n a l com-munication) . Presumably the o l d e r p r e p a r a t i o n s c o n t a i n g r e a t e r numbers of charged groups. Whereas e x c e l l e n t p u r i f i c a t i o n s of r a t i n t e s t i n a l PDase I I were r e p e a t e d l y obtained w i t h the procedure o u t l i n e d above, very poor r e s u l t s were ob t a i n e d w i t h guinea p i g p r e p a r a t i o n s . T h i s was probably due t o the d i f f i c u l t y i n o b t a i n i n g completely s o l u b l e guinea p i g p r e p a r a t i o n s from the s u b c e l l u l a r p a r t i c l e s where the enzyme i s l o c a t e d . 131 I t i s i n t e r e s t i n g t h a t R a z z e l l & Khorana (1961) who used Hllmoe&s" (I960) method (acetone p r e c i p i t a t i o n , ('.NH'u ] 2S'O^ p r e c i p i t a t i o n and aluminium hydroxide g e l adsorption) t o p u r i f y c a l f spleen PDase I I found i t necessary to add amounts of Tween 80, a n o n - i o n i c detergent, d u r i n g s e v e r a l of the steps presumably to i n c r e a s e s o l u b i l i t y of the enzyme. In a d d i t i o n those authors r e p o r t e d t h a t attempts to p u r i f y the enzyme by column chromatography r e s u l t e d i n l o s s e s of a c t i v i t y which they a s c r i b e d to " s u r f a c e or chemical d e n a t u r a t i o n on the s o l i d phase". Despite t h i s B e r n a r d i ' s group have r e p o r t e d no s p e c i a l d i f f i c u l t i e s i n the p u r i f i c a t i o n of hog spleen PDase I I by chromatography on CM-Sephadex, Sephadex G-7 5 and h y d r o x y a p a t i t e (Bernardi & B e r n a r d i , 1968). Perhaps there are s u b t l e d i f f e r e n c e s i n the enzymes from these s p e c i e s which compare w i t h d i f f e r e n c e s r e p o r t e d here between r a t and guinea p i g i n t e s t i n a l PDase I I . Comparison of p a r t i a l l y p u r i f i e d r a t and guinea p i g i n t e s t i n a l PDase I I Because the enzymes were not p u r i f i e d t o the same extent such a comparison i s probably not l e g i t i m a t e s i n c e the guinea p i g PDase was probably more contaminated w i t h other p r o t e i n s which c o u l d complicate the p i c t u r e . For example i t was found t h a t the temperature needed to i n a c t i v a t e 132 the r a t enzyme by 50% rose from 54 WC t o 65^C as the enzyme was p u r i f i e d whereas the corresponding temperatures f o r the guinea p i g enzyme were unchanged (6©°C). I t c o u l d be argued t h a t d u r i n g p u r i f i c a t i o n of the r a t enzyme a substance, p o s s i b l y a pr o t e a s e , was removed and t h i s r e s u l t e d i n an apparent i n c r e a s e i n the t h e r m o s t a b i l i t y of the enzyme. The l a c k of such an e f f e c t i n the case of the guinea p i g c o u l d thus be e x p l a i n e d by the poorer p u r i f i c a t i o n o b tained. However w h i l e some of the other p r o p e r t i e s i n v e s t -i g a t e d were s l i g h t l y d i f f e r e n t , the g e n e r a l impression i s t h a t the enzymes from guinea p i g and r a t were q u i t e s i m i l a r . Both enzymes were i n h i b i t e d by s o l u t i o n s of hi g h i o n i c s t r e n g t h , e x h i b i t e d pH optima i n the 6-7 range and had Km -4 va l u e s of 2-4 x 10 M f o r b i n d i n g to dTpDNP. The a c t i v a t i o n e n e r g i e s of the enzymes";also i n v i t e comparison. In both cases a t r a n s i t i o n was observed w i t h each enzyme d i s p l a y i n g a lower a c t i v a t i o n energy at higher temperatures. Dixon & Webb (1965) have suggested a number of e x p l a n a t i o n s f o r such d i s c o n t i n u i t i e s . These i n c l u d e a) a phase change i n the s o l v e n t ; b) two p a r a l l e l r e a c t i o n s e.g. by d i f f e r e n t activity/ c e n t r e s w i t h d i f f e r e n t temperature c o e f f i c i e n t s ; c) an o v e r a l l process i n v o l v i n g two s u c c e s s i v e r e a c t i o n s w i t h d i f f e r e n t temperature co-e f f i c i e n t s ; d) enzyme e x i s t i n g i n two forms of d i f f e r e n t a c t i v i t i e s and e) r e v e r s i b l e i n a c t i v a t f o n of enzyme. 133 However the study of a number of enzyme systems i n which, the s t r a i g h t l i n e s of d i s c o n t i n u o u s A r r h e n i u s p l o t s do not i n t e r s e c t at the t r a n s i t i o n temperature had l e d Kumamoto e t a l . (1971) t o propose t h a t two independent processes are r e q u i r e d to produce a d i s c o n t i n u i t y and furthermore t h a t the system must al l o w f o r the e x c l u s i v e f u n c t i o n i n g o f each process i n i t s r e s p e c t i v e temperature range. These p r o p o s a l s can be j u s t i f i e d thermodynamically by assuming t h a t the system undergoes a phase change at the c r i t i c a l temperature. While no f i r m data on the s t r u c t u r e of r a t and guinea p i g PDase are a v a i l a b l e some evidence has been d i s c u s s e d i n t h i s t h e s i s t h a t these enzymes can e x i s t i n aggregated or i n s o l u b l e forms under some co n d i t o n s . I t would be i n t e r e s t i n g t o know i f l i p i d i n t e r a c t i o n s ^ a r e i n v o l v e d i n the t r a n s i t i o n but t h i s i s probably u n l i k e l y i n view of the f a c t t h a t the t r a n s i t i o n temperature i n the present case (30°C-40°C) i s much higher than the v a l u e s u s u a l l y a s s o c i a t e d w i t h l i p i d phase t r a n s i t i o n s (see Raison (1973) ) Perhapstthe l a s t word on the i n t e r p r e t a t i o n of such A r r h e n i u s p l o t s should be t h a t of B u l l (1971) who noted "the l o g a r i t h m of the v e l o c i t y of the c r e e p i n g of ants has been p l o t t e d a g a i n s t the r e c i p r o c a l of the absolute temp-e r a t u r e and a l i n e a r r e l a t i o n found w i t h a d i s t i n c t break. 134 i n the s l o p e . At lower temperatures the energy of a c t i v a t i o n c a l c u l a t e d was 25,900cal/mol and at the higher temperatures 12,200cal/mol. Such phenomena are f a r too complex to be i n t e r p r e t e d i n a simple way"! In summary then, the p u r i f i c a t i o n a n d . p r o p e r t i e s o f PDase I I from r a t and guinea p i g i n t e s t i n e have been compared. The g r e a t e r p u r i f i c a t i o n of the r a t enzyme was probably due t o i t s g r e a t e r s o l u b i l i t y s i n c e the guinea p i g enzyme wasjsshown to be r e l a t i v e l y i n s o l u b l e i n a number o f the p u r i f i c a t i o n s t e p s . Both p a r t i a l l y p u r i f i e d enzymes had g r o s s l y s i m i l a r p r o p e r t i e s . PART C. A STUDY OF THE PROPERTIES OF RAT INTESTINAL PDASE II Although s t u d i e s on spleen PDase II by R a z z e l l & Khorana (1961) and B e r n a r d i & B e r n a r d i (1968) and on t e s t i s PDase II by Menon & Smith (1969) have e l u c i d a t e d the b a s i c p r o p e r t i e s and stepwise 5'-*-3' mode o f a c t i o n of the enzyme, l i t t l e i s known about the nature of the enzyme p r o t e i n i t s e l f or about the mechanism by which i t c a r r i e s out the h y d r o l y s i s of t e r m i n a l phosphodiester bonds. Information on these s u b j e c t s would be v a l u a b l e i n comparing t h i s enzyme t o other very w e l l c h a r a c t e r i z e d n u c l e a s e s , such as p a n c r e a t i c RNAase (Barnard, 1969a; Moore & S t e i n , 197 3), DNAase I (Laskowski, 1971; Moore & 135 S t e i n , 1973) , DNAase IT (B e r n a r d i , 19.71) and s t a p h y l o c o c c a l nuclease (Anfinsen e t a l . , 1971). T h e r e f o r e , s i n c e , as d e s c r i b e d i n P a r t B of t h i s t h e s i s , r a t i n t e s t i n a l PDase I I was p u r i f i e d over 500-f o l d , i t was of i n t e r e s t t o study the p r o p e r t i e s o f t h i s h i g h l y p u r i f i e d enzyme i n some d e t a i l . The h y d r o l y s i s o f dTpDNP F i g . 32 shows t h a t when r e l a t i v e l y h i g h amounts o f p u r i f i e d PDase were assayed the p r o d u c t i o n of DNP was p r o p o r t i o n a l to the time of r e a c t i o n f o r o n l y an i n i t i a l p e r i o d ; a f t e r 8-10min a c o n s i d e r a b l e f a l l - o f f i n the r e l e a s e o f DNP was apparent. The n o n - l i n e a r i t y o f the response was not improved by i n c r e a s i n g the dTpDNP con-c e n t r a t i o n , so i t was not the r e s u l t o f s u b s t r a t e d e p l e t i o n , but probably was due to e i t h e r (a) enzyme i n a c t i v a t i o n or more l i k e l y (b) product i n h i b i t i o n o f the r e a c t i o n . Since Raaz-e.ll & Khorana (1961) and F i e r s & Khorana (1963) have r e p o r t e d t h a t spleen PDase I I and L. a c i d o p h i l u s PDase I I r e s p e c t i v e l y were i n h i b i t e d by dTp, i t was p o s s i b l e t h a t t h i s n u c l e o t i d e was r e s p o n s i b l e f o r the f a l l - o f f i n r e a c t i o n r a t e seen i n the presen t case. Experiments which pr o v i d e evidence t h a t t h i s i s so w i l l be d i s c u s s e d l a t e r . In any event the o v e r a l l n o n - l i n e a r i t y o f the h y d r o l y s i s r e a c t i o n with, time d i d not p r e c l u d e measurement of the r a t e s i n c e as^ 136 min F i g . 32: The r e l e a s e of DNP from dTpDNP by p u r i f i e d PDase I I . An a g a r o s e - p u r i f i e d p r e p a r a t i o n whose s p e c i f i c a c t i v -i t y was 430 was employed i n the experiments. A por-t i o n (0.1ml) c o n t a i n i n g 0.9yg of p r o t e i n was added to the PDase I I assay medium (0.9ml) and the p r o d u c t i o n of DNP measured c o n t i n u o u s l y i n a spectrophotometer c u v e t t e a t 37 C. The hatched l i n e r e p r e s e n t s the i n i t i a l r a t e of the r e a c t i o n . 137 F i g . 33 shows t h e i n i t i a l v e l o c i t y o f t h e r e a c t i o n was p r o p o r t i o n a l t o t h e amount o f enzyme p r e s e n t . pH r e q u i r e m e n t s T h e s e h ave b e e n d i s c u s s e d i n some d e t a i l i n p r e v i o u s s e c t i o n s and w i l l o n l y be b r i e f l y m e n t i o n e d h e r e . T a b l e XIX shows t h a t t h e enzyme e x h i b i t s a b r o a d pH optimum a r o u n d pH 6.5 i n a v a r i e t y o f b u f f e r s . The a c t i v a t i o n o b s e r v e d i n i m i d a z o l e b u f f e r s u g g e s t s t h a t p e r h a p s t h i s medium s h o u l d be r o u t i n e l y u s e d t o a s s a y t h e enzyme. The i n h i b i t i o n by p h o s p h a t e was a l s o d e m o n s t r a t e d i n P a r t B. F i g . 34 shows t h e e f f e c t o f a number o f compounds on t h e p H - a c t i v i t y r e l a t i o n s h i p d i s p l a y e d by PDase I I i n i m i d a z o l e - H C l b u f f e r . H i l m o e (I960) and B e r n a r d i & B e r n a r d i 2+ (1968) r e p o r t e d t h a t t h e p r e s e n c e o f Mg c a u s e d a s h i f t t o a l o w e r pH optimum f o r s p l e e n PDase I I . No s u c h s s h i f t 2+ was o b s e r v e d f o r i n t e s t i n a l PDase I I w i t h Mg o r w i t h EDTA. The s l i g h t i n h i b i t i o n by (NH it) 2 SO if has been d i s c u s s e d i n P a r t B. M o l e c u l a r w e i g h t T h i s was d e t e r m i n e d by g e l - f i l t r a t i o n -using tyro d i f f e r e n t g e l s y s t e m s ( F i g . 3 5 ) . By u s i n g t h e method o f L a u r e n t & K i l l a n d e r (1964). a m o l e c u l a r w e i g h t o f 155,.Q.0Q,. 138 of protein o 1.0 2.0 91 1 1 1 r pi of enzyme soln. F i g . 33: The a c t i v i t y o f r a t PDase I I as a f u n c t i o n o f t h e amount o f enzyme p r e s e n t . The s p e c i f i c a c t i v i t y o f t h e p r e p a r a -t i o n was a b o u t 200. D i f f e r e n t amounts o f t h e p r e p a r a t i o n w e r e a s s a y e d f o r PDase I I u s i n g M e t h o d I . 139 Tabl e XIX. PDase I I a c t i v i t y i n a v a r i e t y o f b u f f e r s . PDase IT 'CO.. Q7units ] L was assayed as d e s c r i b e d i n M a t e r i a l s and Methods s e c t i o n except t h a t the B u f f e r Cat a c o n c e n t r a t i o n o f 0.IMI was v a r i e d as i n d i c a t e d . B u f f e r Optimum pK A c t i v i t y C%1 sodium s u c c i n a t e 6.10-6.55 100 ME'S-NaOH 6.55 103 Imidazole-HCl 6.60 12 0 sodium phosphate 6.70. 73 140 i r - r 501 . 1 • 1 1 1 5 6 7 8 pH F i g . 34: E f f e c t o f M g C l 2 , EDTA and ( N H 4 ) 2 S 0 4 on the pH optimum o f r a t PDase I I . An agarose p u r i f i e d enzyme p r e p a r a t i o n was used. Aligiuiots ( c o n t i n i n g 0. 5yg of p r o t e i n ) were assayed f o r PDase I I by Method I except t h a t the approp-r i a t e a d d i t i o n s were made and the b u f f e r was 0.1M : i m i d a z o l e a d j u s t e d t o the i n d i c a t e d pH w i t h HCly The a d d i t i o n s were: o, none; • , lOmMtfi EDTA; A, l O m M M g C l 2 ; A , lOOmMN'' (NH4) 2 S 0 4 . 141 1.0 0.8 0.6 > O 0.4 0.2 i—I—l I I I T 1 1 1—I I I I _I_L ioH 10 Mol.Wt. 10 F i g . 3 5 : M o l e c u l a r weight e s t i m a t i o n o f r a t i n t e s t i n a l PDase II by g e l f i l t r a t i o n chromatography. The experiments were c a r r i e d out as d e s c r i b e d i n the M a t e r i a l s and Methods s e c t i o n , u s i n g B i o - G e l A-0.5m (hatched curve) and Sephadex G-150 s u p e r f i n e ( s o l i d c u r v e ) ; the e l u t -i n g b u f f e r s were 50mM sodium acetate-0.5M NaCl pH 5 and 20mM sodium phosphate-0.2M NaCl pH 7.2 r e s p e c t i v e -l y . The p r o t e i n markers used were: A, cytochrome c (mol. wt. 12400); V, RNAse A (mol. wt. 13700); • , myoglobin (mol. wt. 17800); v, chymo-t r y p s i n o g e n A (mol. wt. 25000); • , ovalbumin (mol. wt. 45000); , E. c o l i a l k a l i n e phosphatase (mol. wt. 8 0000); O/ ye a s t a l c o h o l dehydrogenase (mol. wt. 151000); A , a l d o l a s e (mol. wt. 158000); ©, ' c a t a l a s e (mol. wt. 1 9 5 0 0 0 1 ) ; O, a p o f e r r i t i n (mol. wt. 480000). The K a v v a l u e s o f PDase I I are i n d i c a t e d by the arrows. 1 Although the t r u e mol. wt. of c a t a l a s e i s i n the r e g i o n o f 240000 , Andrews (1965) has shown t h a t the enzyme behaves as a p r o t e i n w i t h a mol. wt. of 195000 i n g e l f i l t r a t i o n experiments. 142 was obtained with Bio-Gel A-0.5m whereas a value of 170,000 was deduced from experiments on Sephadex G-150. The only other value reported for an enzyme of t h i s type was 100,000 for t e s t i s PDase II by Menon & Smith .'(1969). Van Venrooj & Poort (1970) showed that pancreatic PDase II was eluted from a Sephadex G-100 column just after the void volume whereas Bernardi & Bernardi (1968) reported that the sedimentation constant of spleen PDase was 4.6S which would correspond to a molecular weight of 4 6.0-6.5 x 10 (Schachman, 1959). Thus i t would appear that rat i n t e s t i n a l PDase II i s considerably larger than the enzymes from other sources. I s o e l e c t r i c point This was investigated by i s o e l e c t r i c focusing, a procedure developed by Vesterberg & Svensson (1966) to separate proteins (and other ampholytes) according to t h e i r d i f f e r e n t i s o e l e c t r i c points. In p r i n c i p l e , the method i s quite similar to electrophoresis but d i f f e r s from i t i n that i t i s not car r i e d out at a s p e c i f i c pH value. Rather a gradient of pH i s used and t h i s i s accomplished by using synthetic polyamino-polycarboxylic acids wh£ch possess subtly d i f f e r e n t i s o e l e c t r i c points. In an e l e c t r i c f i e l d these c a r r i e r ampholytes (AmpholineL quickly form a gradient of pH. from anode to cathode on 143 which, a mixture of p r o t e i n s can be separated. The added p r o t e i n s move towards th e anode or cathode a c c o r d i n g to t h e i r net charge but e v e n t u a l l y become s t a t i o n a r y at a p o i n t i n the pH g r a d i e n t equal to t h e i r r e s p e c t i v e i s o -e l e c t r i c p o i n t s . The r e s u l t s o f two i s o e l e c t r i c f o c u s i n g e x p e r i -ments are shown i n F i g . 36. In the f i r s t ( F i g . 36A) a pH g r a d i e n t of 3-10 was used and the peak of PDase I I a c t i v i t y was found a t pH 4.2. However when a narrower pH range was chosen the p a t t e r n of PDase a c t i v i t y was more complex. ( F i g . 36B). Although a peak o f a c t i v i t y was found a t pH 4.5, a v a l u e c l o s e to t h a t observed i n the f i r s t experiment, most o f the recovered PDase was found at the extremes o f the pH g r a d i e n t at pH 3.4 and a t pH 7.2. Salmon t e s t i s PDase has been r e p o r t e d by Menon & Smith (1969) to have an i s o e l e c t r i c p o i n t of 7.0. Gel e l e c t r o p h o r e s i s T h i s was c a r r i e d out a t pH 8.5 u s i n g 5% p o l y -acrylamide g e l s . With f r e s h p r e p a r a t i o n s o f p u r i f i e d r a t i n t e s t i n a l PDase I I the r e s u l t s shown i n F i g . 37 were o b t a i n e d . S t a i n i n g a g e l w i t h Coomassie Blue gave r i s e to 8-10 bands whereas when an i d e n t i c a l g e l was s l i c e d and each s l i c e analyzed f o r PDase I I a c t i v i t y , most o f the enzyme a c t i v i t y seemed to be a s s o c i a t e d w i t h one o f the 144 fraction no. F i g . 36: E l e c t r o f o c u s i n g o f p u r i f i e d r a t i n t e s t i n a l PDase I I . Agarose p u r i f i e d p r e p a r a t i o n s were used. In A pH 3-10 c a r r i e r ampholytes were used and the experiment was c a r r i e d on f o r 31 h, whereas i n B pH 3-5 c a r r i e r ampho-l y t e s were employed and the e l e c t r o f o c u s i n g p e r i o d was 90 h. The recover y of PDase I I a c t i v i t y i n A and B was 75% and 11% r e s p e c t i v e l y . •, PDase I I ; », pH. 145 F i g . 37: Polyacrylamide g e l e l e c t r o p h o r e s i s of f r e s h l y p u r i f i e d r a t PDase I I . 5% g e l s were prepared and run as d e s c r i b e d i n the M a t e r i a l s and Methods s e c t i o n . Samples of an a g a r o s e - p u r i f i e d f r a c t i o n (about 50yg of p r o t e i n each) were a p p l i e d to two i d e n t i c a l g e l s . A f t e r e l e c t r o p h o r e s i s one g e l was s t a i n e d f o r p r o t e i n e s t i m a t i o n with Coomassie Blue w h i l e the other was s l i c e d i n 2 mm p i e c e s which were then e x t r a c t e d and assayed f o r PDase I I a c t i v i t y (•) as d e s c r i b e d i n the Methods s e c t i o n . The s t a i n e d g e l was scanned a t 500 nm i n a G i l f o r d spectrophotometer equipped w i t h a l i n e a r t r a n s p o r t d e v i c e ( s o l i d c u r v e ) ; a photograph of the s t a i n e d g e l i s a l s o shown. 146 l e s s i n t e n s e p r o t e i n bands. A s m a l l e r , slower-moving band o f PDase a c t i v i t y was a l s o seen. Menon & Smith Q.9.69.1 a l s o r e p o r t e d t h a t t e s t i s PDase I I c o u l d be r e s -o l v e d e l e c t r o p h o r e t i c a l l y i n t o two p r o t e i n components both of which e x h i b i t e d enzyme a c t i v i t y . A number o f experiments were a l s o c a r r i e d out w i t h a p u r i f i e d PDase p r e p a r a t i o n which had been "aged" as d e s c r i b e d i n the M a t e r i a l s and Methods s e c t i o n . T h i s p r e p a r a t i o n gave very d i f f e r e n t r e s u l t s from those d e s c r i b e d above, as i l l u s t r a t e d i n F i g . 38. Only a s i n g l e d i f f u s e p r o t e i n band was observed and t h i s co-i n c i d e d w i t h the enzyme a c t i v i t y on the g e l . No carbo-hydrate m a t e r i a l was d e t e c t a b l e i n the band. Moreover when the e l e c t r o p h o r e s i s was performed i n the presence of 4M urea, the p a t t e r n was s i m i l a r although now the c o i n c i d e n t bands of a c t i v i t y and .protein were found to have migrated more q u i c k l y towards the anode ( F i g . 38). A p o s s i b l e e x p l a n a t i o n f o r these r e s u l t s would be t h a t aggregation was r e s p o n s i b l e f o r the many p r o t e i n bands observed w i t h the " f r e s h " p r e p a r a t i o n s w h i l e the "aging" process a c c e l e r a t e d the breakdown o f these aggregates. However i f t h i s was so, one might have expected the PDase I I a c t i v i t y t o be a s s o c i a t e d w i t h each o f the p r o t e i n bands i n the f i r s t case. The r e s u l t s might a l s o be e x p l a i n e d by the presence of a weak protease a c t i v i t y i n the p u r i f i e d p r e p a r a t i o n s of PDase TT. Some 147 F i g . 38: P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s o f a p u r i f i e d p r e p -a r a t i o n o f r a t PDase I I w h i c h h a d b e e n " a g e d " . The " a g i n g " , e l e c t r o p h o r e s i s a n d s t a i n i n g ( C o o m a s s i e B l u e ) p r o c e d u r e s a r e d e s c r i b e d i n t h e M a t e r i a l s a n d M e t h o d s s e c t i o n . The s a m p l e i n e a c h c a s e c o n t a i n e d 26ug o f p r o t e i n and t h e e l e c t r o p h o r e s i s was c a r r i e d o u t i n t h e a b s e n c e ( l e f t ) a n d p r e s e n c e ( r i g h t ) o f 4 M u r e a . 148 evidence t h a t such an a c t i v i t y i s indeed p r e s e n t i n r a t p r e p a r a t i o n s has Been d i s c u s s e d i n P a r t s A and B. The l a t t e r s p e c u l a t i o n i s supported by the f o l l o w i n g obser-v a t i o n s : (a) d u r i n g e l e c t r o p h o r e s i s of the "aged" p r e p a r a t i o n s ( F i g . 38) s m a l l amounts o f s t a i n a b l e m a t e r i a l were observed to c o - e l e c t r o p h o r e s e w i t h the bromophenol blue which marked the " f r o n t " . T h i s low m o l e c u l a r -weight substance c o u l d be be the h y d r o l y s i s product of a protease; (b) "aged" p r e p a r a t i o n s o f PDase were i n v a r -i a b l y found to e x h i b i t lower s p e c i f i c a c t i v i t i e s C2 00-300units/mg of p r o t e i n ) compared to f r e s h p r e p a r a t i o n s (300-500units/mg of p r o t e i n ) . The l o s s o f enzyme a c t i v i t y might have been due to p r o t e o l y t i c a c t i o n although i n another s e r i e s of experiments i t was found t h a t the p u r i f i e d enzyme was somewhat r e s i s t a n t t o both pronase and t r y p s i n , a t l e a s t i n a 2h i n c u b a t i o n a t 37°C (Table XX). Contaminating enzyme a c t i v i t i e s The s p e c i f i c a c t i v i t i e s o f a number of p o l y -n u c l e o t i d a s e s and phosphatases p r e s e n t i n crude p r e p a r a t i o n s o f r a t i n t e s t i n a l mucosa a r e shown i n Table XXI. The same a c t i v i t i e s were determined i n the p u r i f i e d p r e p a r a t i o n s but b e s i d e s PDase I I o n l y DNAase I I and RNAase I I a c t i v i t i e s were d e t e c t a b l e . The enrichment of t h e i r s p e c i f i c a c t i v i t i e s was much lower than t h a t observed f o r PDase 3;T. 149 T a b l e .XX. E f f e c t o f pronase and t r y p s i n on PDase II a c t i v i t y . P u r i f i e d PDase IT CQ.7unitsI was incubated f o r the times i n d i c a t e d at 37°C w i t h pronase or t r y p s i n as d e s c r i b e d by Henning e t a l . CT973X; the p r o t e o l y t i c enzyme c o n c e n t r a t i o n was -TOOyg/ml. Treatment PDase II a c t i v i t y remaining C%1 None 100 pronase C2h) 20 pronase Cl 2 h i 0 t r y p s i n (2hl 47 t r y p s i n (12h) 0 150 T a b l e XXI. Contaminating enzyme a c t i v i t e s i n p u r i f i e d preparations- of r a t i n t e s t i n a l PDase IT. PDase I I was p u r i f i e d from a crude e x t r a c t as d e s c r i b e d i n the M a t e r i a l s and Methods s e c t i o n . The a c t i v i t i e s of a number of enzymes were determined i n the crude and p u r i f i e d f r a c t i o n s and, where p o s s i b l e , the enrichment of a c t i v i t y ( s p e c i f i c a c t i v i t y i n the p u r i f i e d f r a c t i o n / s p e c i f i c a c t i v i t y i n the crude f r a c t i o n ! was c a l c u l a t e d . S p e c i f i c a c t i v i t y Enzyme a c t i v i t y i n crude f r a c t i o n Enrichment (units/mg) PDase I I 0.77 540 PDase I 0.61 0 DNAase I 0.10 0 DNAase I I 0.08 7.8 a c i d i c RNAase I I 4.0 27.6 a c i d i c phosphatase 0.97 0 a l k a l i n e phosphatase 0.89 0 5 1 - n u c l e o t i d a s e 1.4 0 adenosine deaminase 4.8 0 151 I t i s n o t known i f t h e s e a c t i v i t i e s r e p r e s e n t t h e p r e s e n c e o f c o n t a m i n a t i n g enzymes i n t h e p u r i f i e d p r e p a r a t i o n o r i f i n f a c t t h e y a r e due t o t h e i n t r i n s i c p o l y n u c l e o t i d a s e a c t i v i t y o f t h e PDase I I . H y d r o l y s i s o f p h o s p h o d i e s t e r s The p a p e r c h r o m a t o g r a m on w h i c h t h e h y d r o l y s i s o f a d i n u c l e o s i d e m o n o p h o s p h a t e , GpA, b y p u r i f i e d P Dase I I was f o l l o w e d i s i l l u s t r a t e d i n F i g . 39. The p r o d u c t s , a s e x p e c t e d , w e r e g u a n o s i n e 3 ' - p h o s p h a t e and a d e n o s i n e and w e r e f o r m e d i n e q u i m o l a r a m o u n t s . They w e r e i d e n t -i f i e d b y t h e u s e o f c h r o m a t o g r a p h i c s t a n d a r d s a n d b y t h e i r r e s p e c t i v e u v s p e c t r a . The i d e n t i t y o f Gp was c o n f i r m e d b y i t s r e s i s t a n c e t o t h e 5 ' - n u c l e o t i d a s e i n c r u d e s n a k e venom ( s e e t h e M a t e r i a l s and M e t h o d s s e c t i o n ) . S i m i l a r r e s u l t s w e r e o b t a i n e d w i t h ApG w h i c h was h y d r o l y s e d t o Ap and G, b u t a s shown i n T a b l e X X I I , b o t h t h e s e d i n u c l e o s i d e m o n o p h o s p h a t e s w e r e c l e a v e d a t much s l o w e r r a t e s t h a n was dTpDNP. P o l y A was h y d r o l y s e d o n l y a t a v e r y s l o w r a t e by PDase I I and t h e o n l y p r o d u c t d e t e c t e d by p a p e r c h r o m a t o g r a p h y was Ap ( T a b l e X X I I ) . The h y d r o l y s i s b y p u r i f i e d P Dase I I o f a number o f o t h e r compounds c o n t a i n i n g p h o s p h o d i e s t e r b o n d s i s a l s o s u m m a r i z e d by t h e d a t a i n T a b l e X X I I . B o t h t h e n i t r o p h e n y l and d i n i t r o p h e n y l e s t e r s o f t h y m i d i n e 3 ' - p h o s p h a t e a l o n g 152 1.00 0.75 OH 0.50 0.25 0 (38) J88^ 0088 -000 JL _L 0 1 2 3 hours A G p A G p F i g . 39: The , h y d r o l y s i s o f GpA by p u r i f i e d r a t PDase I I . GpA (liiomofil) was incubated w i t h PDase I I (0.9 u n i t s ) i n 0. IMM. sodium a c e t a t e b u f f e r pH 6 (0. 3 mliX) at 37°C and a l i q u o t s (50ttslL) were removed a f t e r 0, 1, 2 and 3hh had elap s e d . These were quenched w i t h 5ptjlT o f g l a c i a l a c e t i c a c i d and a p p l i e d t o a sheet of Whatman No. 40 papers Chromatography was c a r r i e d out f o r 30hh i n s o l v e n t C and the IJy absorbing spots were c u t out, e l u t e d as d e s c r i b e d i n the Methods s e c t i o n and i d e n t i f i e d and c h a r a c t e r i z e d as s t a t e d i n the t e x t . The f i g u r e i s a drawing of the chromatogram and the numbers i n each spot r e f e r t o the amounts of m a t e r i a l p r e s e n t ( i n nanomoles). 153 T a b l e XXII. The r a t e of h y d r o l y s i s o f some compounds c o n t a i n i n g phosphodiester bonds by p u r i f i e d PDase IT. PDase TT CQ.8units i n e x p t . l and 0.9units i n expt. 2). was incubated w i t h each compound i n 0.1M sodium s u c c i n a t e b u f f e r pH 6.1 o r , i n the case of GpA, ApG and p o l y A i n 0.IM sodium a c e t a t e b u f f e r pH 6. The f i n a l c o n c e n t r a t i o n of each- compound was ImM except f o r RNA "core" (0. 5mg/ml) and p o l y A (lmg/ml). The r a t e . o f h y d r o l y s i s o f dTpDNP, dTpNP, b i s - ( p - n i t r o p h e n y l j phosphate and NPpdT was determined s p e c t r o p h o t o m e t r i c a l l y ; t h a t of GpA, ApG and p o l y A by paper chromatographic e s t i m a t i o n i n s o l v e n t C o f the adenosine, guanosine or Ap formed r e s p e c t i v e l y from these compounds; t h a t o f ATP by. measuring the P^ formed. Rate o f H y d r o l y s i s (umol/h/mg of p r o t e i n ) Compound Expt.1 Expt.2 dTpDNP 311 270 dTpNP 213 RNA "core" 4 90V1 GpA (3*+5') 130 66 GpA (2,-*5') <10 ApG (3'+5') - 39 bi s - C p - n i t r o p h e n y l ) phosphate 1.1 NPpdT < 0.1 ATP < 0.5 p o l y A - 15 *" assuming an average e v a l u e o f 10,600 a t 260nm 154 w i t h RNA "core" and GpA were h y d r o l y s e d by the enzyme, though at s l i g h t l y d i f f e r e n t r a t e s . RNA "core" i s the non-d i f f u s i b l e m a t e r i a l remaining a f t e r d i a l y s i n g the o l i g o -n u c l e o t i d e products of exhaustive RNAase a c t i o n , and i n the PDase II s u b s t r a t e o r i g i n a l l y used by Hilmoe (1960) . In view of the present r e s u l t s o b tained w i t h t h i s s u b s t r a t e , i t i s s u r p r i s i n g t h a t i n an e a r l i e r r e p o r t , Hilmoe & Heppel (1955) showed t h a t a p a r t i a l l y p u r i f i e d " i n t e s t i n a l PDase" was i n a c t i v e towards RNA "core". One must assume t h a t d i f f e r e n t a c t i v i t i e s were being examined i n each case. I t i s i n t e r e s t i n g t o note t h a t a d e n y l y l - (5 *-»-2 1) guanosine was not a s u b s t r a t e f o r the enzyme and t h a t b i s - ( p -n i t r o p h e n y l ) phosphate was h y d r o l y s e d at l e s s than 0.5% the r a t e of h y d r o l y s i s of dTpDNP. Although B e r n a r d i & B e r n a r d i (1968) r e p o r t e d t h a t b i s - ( p - n i t r o p h e n y l ) phosphate was "a r a t h e r poor s u b s t r a t e " f o r spleen PDase they gave no data t h a t might be compared wi t h the p r e s e n t case. However, i t i s e v i d e n t t h a t the s o - c a l l e d n o n - s p e c i f i c PDase (Swenson & Hodes 1969; S l o r 1970; S i c a r d e t a l . 1970) which h y d r o l y s e s b i s - ( p - n i t r o p h e n y l ) phosphate i s a d i s t i n c t l y d i f f e r e n t enzyme from PDase I I d e s c r i b e d h e r e i n (see under D i s c u s s i o n ) . No h y d r o l y s i s of thymidine 5 ' - ( p - n i t r o p h e n y l ) phosphate c r ATP a t pH 6.1 was d e t e c t a b l e . The former compound i s a s u b s t r a t e f o r PDase I ( R a z z e l l , 1961; Hynie & Zbarsky, 1970a, hi w h i l e the l a t t e r n u c l e o t i d e iis a 155 .substrate f o r n u c l e o s i d e polyphosphatase (Laskowski & Fl-lipowiez .'(.15958).. In view of the f a c t t h a t of the compounds l i s t e d i n Table XXII 6hl y dTpDNP, dTpNP, GpA, ApG and RNA "core" were h y d r o l y s e d by PDase I I i t might be deduced t h a t o n l y those compounds c o n t a i n i n g a n u c l e o s i d e 3 *-phosphoryl group were capable of being bound a t the a c t i v e c e n t r e o f the enzyme. I t should be mentioned here t h a t no evidence was found i n these experiments t h a t r a t i n t e s t i n a l PDase II i s a b l e to c a r r y out the " t r a n s f e r " r e a c t i o n s which R a z z e l l & Khorana (1961) and Menon & Smith (197 0) have d e s c r i b e d f o r the spleen and t e s t i s enzymes r e s p e c t i v e l y . Even when c o n c e n t r a t i o n s of GpA as h i g h as 8mM were incubated w i t h the r a t enzyme, the o n l y d e t e c t a b l e products were Gp and A. E f f e c t o f phosphodiesters on dTpDNP h y d r o l y s i s Experiments Of t h i s s o r t can g i v e v a l u a b l e i n f o r m a t i o n on the b i n d i n g o f p o s s i b l e s u b s t r a t e s t o an enzyme, s i n c e i t would be expected t h a t a good s u b s t r a t e would c o m p e t i t i v e l y i n h i b i t the h y d r o l y s i s of dTpDNP. The r e s u l t s shown i n Table XXIII bear t h i s out, to some ex t e n t . The d i n u c l e o s i d e monophosphates GpA and ApG i n h i b i t dTpDNP h y d r o l y s i s t o d i f f e r e n t degrees and t h i s -156 T a b l e XXITT. E f f e c t o f pho s phod i e s t er s on the r a t e o f dTpDNP hydro l y s i s : by- p u r i f i e d PDase I I PDase I I CO. 14ainitsl was assayed b~y Method I i n the presence of the compounds shown. A d d i t i o n Concn. (mML PDase IT a c t i v i t y C%)1 None ApG GpA p o l y A NAD ATP glycerop h o s p h o r y l -c h o l i n e 3 ' - , 5 ' - c y c l i c AMP 3 ' - , 5 ' - c y c l i c AMP 1 1 lmg/ml 1 1 1 1 10 10.0. 63 37 111 100 10.0., 95 99 93 157 d i f f e r e n c e might be r e l a t e d to the r a t e s a t which these themselves were h y d r o l y s e d by the enzyme (Table X X I I ) . By the same reaso n i n g one might suppose t h a t NAD, ATP, g l y c e r y l p h o s p h o r y l c h o l i n e and S'^'-cAMP were poor s u b s t r a t e s f o r the enzyme s i n c e they d i d not i n h i b i t enzymatic h y d r o l y s i s o f dTpDNP. T h i s i s noteworthy because PDases which h y d r o l y s e NAD ( F u t a i and Mizuno, 1967? R a z z e l l , 1968), ATP (Laskowski and F i l i p o w i c z , 1958), 3 ' 5 ' - c y c l i c AMP (Drummond & Hamamoto, 1971) and g l y c e r y l -phosphoryl c h o l i n e ( H a y a i s h i , 1955) are known. E v i d e n t l y the s p e c i f i c i t i e s of these enzymes and PDase I I are q u i t e d i f f e r e n t . E f f e c t of phosphomoneesters /These r e s u l t s are shown i n Table XXIV. The most i n h i b i t o r y mononucleotide t e s t e d was dTp, which i n h i b i t e d the enzyme by 62% a t a c o n c e n t r a t i o n of ImM. Deoxy-thymidine 5'-phosphate had almost no e f f e c t . In the adenosine monophosphate s e r i e s Ap was most i n h i b i t o r y . However i t i s i n t e r e s t i n g t h a t one has t o i n c r e a s e the Ap c o n c e n t r a t i o n almost t e n - f o l d i n order to o b t a i n the degree of i n h i b i t i o n seen w i t h ImM Tp. I f as seems l i k e l y from these r e s u l t s , Ap i s not bound v e r y t i g h t l y t o the enzyme, t h i s c o u l d explain' the low degree of b i n d i n g of ApG noted e a r l i e r . The v e r y slow r e l e a s e o f Ap from p o l y A might a l s o be a consequence of t h i s - lower degree of b i n d i n g . 158 T a b l e XXTV. E f f e c t of some phosphomonesters on the rate of dTpDNP Hydrolysis by p u r i f i e d PDase. PDase TT CClQunitsI was assayed by Method I in the presence of the mononucleotides shown. A d d i t i o n Concn. PDase I I a c t i v i t y (mM) C%I None - 100 5'-AMP 1 99 5'-AMP 10 63 3'-AMP 1 92 3'-AMP 10 28 2'-AMP 1 98 2'-AMP 10 47 3'-dTMP 1 38 5'-dTMP 1 96 159 As expected the i n h i b i t i o n by Tp and by GpA Csee above) was found t o be c o m p e t i t i v e ( F i g . 4 0 1 . In a d d i t i o n a Dixon p l o t (Dixon, 1953) o f the i n h i b i t i o n o f PDase by Tp showed ( F i g . 41). the i n h i b i t o r c o n s t a n t (Ki) -5 f o r this,"..nucleotide to be about 2 x 10 M. T h i s v a l u e , which i s of the same order of magnitude as the Km v a l u e ( F i g s . 29 & 30) i n d i c a t e s a st r o n g degree o f i n t e r a c t i o n w i t h the enzyme and e x p l a i n s the sharp f a l l - o f f o f DNP r e l e a s e noted e a r l i e r i n enzyme assays (see F i g . 32). E f f e c t o f d i v a l e n t c a t i o n s Hilmoe (19601 o r i g i n a l l y r e p o r t e d t h a t these ions i n h i b i t spleen PDase I I but Menon & Smith have shown t h a t f o r the salmon t e s t i s enzyme a t l e a s t , severe i n h i b -i t i o n c o u l d o n l y be ob t a i n e d w i t h C u + + . The f i n d i n g s f o r i n t e s t i n a l PDase, shown i n Tab l e XXV, are more i n accord w i t h the l a t t e r p i c t u r e . Mg , Ca , Mn , Co and N i + + a t a c o n c e n t r a t i o n of lOmM were v i r t u a l l y without ++ ++ e f f e c t . In c o n t r a s t , Cu and t o a l e s s e r extent Zn i n h i b i t e d the enzyme a c t i v i t y c o n s i d e r a b l y . As noted p r e v i o u s l y lOOmM EDTA had almost no e f f e c t on the enzyme a c t i v i t y (see Table X V I I ) . E f f e c t o f enzyme i n h i b i t o r s O c c a s i o n a l l y important i n s i g h t s i n t o an enzyme's s t r u c t u r e can be gained by the use of s p e c i f i c enzyme 40: Competitive i n h i b i t i o n o f p u r i f i e d - - r a t PDase I I by GpA and Tp. The experiment was" c a r r i e d out e x a c t l y as d e s c r i b e d i n the legend f o r F i g . 29A except t h a t the f o l -lowing a d d i t i o n s were made: ImM GpA; A, 1 mM Tp; •, 0.1 mM Tp; •/ none. 161 | 1 I I I I 1 0 0.4 0.8 10x[Tp] F i g . 4 1 : Dixon p l o t of the i n h i b i t i o n of PDase I I by Tp. The enzyme p r e p a r a t i o n was t h a t used i n the experiments i l -l u s t r a t e d i n F i g s . 29A and 40. PDase I I a c t i v i t y was assayed by Method I w i t h 0.4 mM (•) or 0.8 mM (•) dTpDNP i n the presence of v a r i a b l e c o n c e n t r a t i o n s of Tp. 162 T a b l e XXV• E f f e c t o f d i v a l e n t c a t i o n s on PDase IT a c t i v i t y . PDase I I CO. l l u n i t s J L was assayed witFL dTpNP u s i n g Method IT i n the presence of the s a l t s shown. A d d i t i o n Concn. PDase I I a c t i v i t y CmMl C%I None 100 MgCl 2 10 9-6 C a C l 2 10 101 MnCl 2 10. 95 C o C l 2 10 9-5 N i C l 2 10. 99 Z n C l 2 10 76 CuS0 4 1 74 CuS0 4 10 34 163 i n h i b i t o r s . Since v i r t u a l l y nothing i s known about the a c t i v e s i t e o r even the gross s t r u c t u r e o f PDase I I i t was thought t h a t such experiments might prove rewarding. The r e s u l t s i n Table XXVI show the e f f e c t s o f a number of enzyme i n h i b i t o r s on PDase a c t i v i t y . N e i t h e r mercaptoethanol, d i t h i o t h r e i t o l , f l u o r i d e nor t h e o p h y l l i n e a f f e c t e d the enzyme a c t i v i t y t o any g r e a t e x t e n t . The former two compounds are re d u c i n g agents whereas the l a t t e r two are potent i n h i b i t o r s f o r a c i d phosphomonoesterase (Hollander, 1971) and c y c l i c n u c l e o t i d e PDase r e s p e c t i v e l y (Drummond & Yamamoto, 1971). T h i o l reagents such as N-ethylmaleimide and o r g a n i c m e r c u r i a l s were q u i t e i n h i b i t o r y as was i o d o a c e t a t e but iodoacetamide was without e f f e c t . The q u e s t i o n arose as t o whether these o b s e r v a t i o n s had a common l i n k o r whether e s s e n t i a l l y d i f f e r e n t f u n c t i o n a l groups on the enzyme were being a f f e c t e d by the v a r i o u s reagents. Since the i n h i b i t i o n by i o d o a c e t a t e was markedly a f f e c t e d by the time o f p r e i n c u b a t i o n w i t h the enzyme whereas the i n h i b i t i o n by PCMPS was not (Table XXVI) i t would appear t h a t d i f f e r e n t p r ocesses were i n v o l v e d . The d i f f e r e n c e s o b t a i n e d w i t h i o d o a c e t a t e and iodoacetamide (Table XXVI) are r e m i n i s c e n t of the r e s u l t s r e p o r t e d f o r RNAase (Gundlack e t al_. , 1959a) , DNAase I ( P r i c e e t a l . , 1969), DNAase I I (Oshima & P r i c e , 19731 and RNAase T 2 (Takahashi e t a l . , 19671. Because PCMPS r e a c t s with: t h i o l groups-, an attempt 164 Table XXVI. E f f e c t of a number o f p o s s i b l e enzyme i n h i b i t o r s on PDase I I a c t i v i t y PDase I I CO. 14units)" was estimated b y IVIefehod I i n the presence of the i n h i b i t o r s shown. Except where noted the enzyme was pr e - i n c u b a t e d w i t h the a d d i t i o n f o r 5min a f t e r which dTpDNP was added to s t a r t the r e a c t i o n . A d d i t i o n Concn. PDase IT a c t i v i t y (mM) m None - 100. potassium f l u o r i d e 5 103 t h e o p h y l l i n e 1 101 II 5 9.3 mercaptoethanol 1 104 II 5 106 d i t h i o t h r e i t o l 5 103 II 10 93 N-ethylmaleimide 5 84 p - c h l o r o m e r c u r i -benzoate 1 30, p - c h l o r o m e r c u r i -pheny1sulphonate II 1 62 5 37 4 II 5 37 2 II 5 31? i o d o a c e t a t e 1 86 II 5 23 II 5 0* iodoacetamide 1 9.9 II 5 97 II 5 9.8 s p r e - i n c u b a t e d f o r 15s i n s t e a d o f 5min pre - i n c u b a t e d f o r 30min i n s t e a d of 5min 165 was made t o a l l a y i t s e f f e c t by- the a d d i t i o n o f mercapto-eth a n o l ( F i g . 4 2 1 . S u r p r i s i n g l y , q u i t e the o p p o s i t e r e s u l t was o b t a i n e d . The a d d i t i o n o f the redu c i n g agent i n the presence o f PCMPS induced a p r o g r e s s i v e decay o f the enzyme a c t i v i t y . P o s s i b i l y the t e r t i a r y s t r u c t u r e of the enzyme was d i s r u p t e d s u f f i c i e n t l y by the mercaptoethanol t o a l l o w the m e r c u r i a l t o r e a c t w i t h -SH groups normally b u r i e d i n the enzymerrmolecule. The d i f f e r i n g e f f e c t s of i o d o a c e t a t e and i o d o -acetamide were i n v e s t i g a t e d more c l o s e l y by i n c u b a t i n g some PDase wit h the compounds and a s s a y i n g a l i q u o t s removed from the mixture a t v a r i o u s times f o r r e s i d u a l PDase a c t i v i t y . While i t was again observed t h a t iodoacetamide had a b s o l u t e l y no e f f e c t , the i n a c t i v a t i o n r a t e i n i o d o -a c e t a t e was seen t o f o l l o w approximately f i r s t o r d e r k i n e t i c s ( F i g . 43); evidence t h a t more than one r e a c t i n g s p e c i e s was presen t can be i n f e r r e d from the n o n - l i n e a r nature o f the curve. Approximately 21% of the PDase a c t i v i t y was i n a c t i v a t e d at a s l i g h t l y f a s t e r r a t e than the remaining 79%. F i g u r e 44 shows t h a t the i n a c t i v a t i o n o f PDase by io d o a c e t a t e was a l s o pH-dependent. In the presence o f 5mM io d o a c e t a t e the enzyme had a h a l f - l i f e of about 160min at pH 9 but o n l y 20min a t pH 6.1 or lower. T h i s obser-v a t i o n w i l l be d i s c u s s e d l a t e r . 166 F i g . 42: C u m u l a t i v e e f f e c t o f PCMPS a n d m e r c a p t o e t h a n o l on r a t PDase I I a c t i v i t y . PDase I I (8 u n i t s ) was i n c u b a t e d a t 24°C i n 0.1 M s o d i u m s u c c i n a t e pH 6.1 (1.0 ml) i n t h e p r e s e n c e o f t h e f o l l o w i n g a d d i t i o n s : •, n o n e ; A, 12.5 mM m e r c a p t a e t h a n o l ; •, 12.5 mM PCMPS; and •, 12.5 mM m e r c a p t o e t h e n o l - 12.5 mM'PCMPS. S a m p l e s (0.1 ml) w e r e r e m o v e d a t i n t e r v a l s a n d a s s a y e d f o r PDase I I a c t i v i t y u s i n g M e t h o d I . The a c t i v i t y i s e x p r e s s e d a s a p e r c e n t a g e o f t h e i n i t i a l v a l u e ancTthe h a t c h e d l i n e r e p r e s e n t s u n c h a n g e d PDase I I a c t i v i t y . 167 F i g . 43: I n a c t i v a t i o n of PDase I I by i o d o a c e t i c a c i d . P u r i f i e d PDase I I (8.4 u n i t s ) was incubated a t 24oc i n 0.1 M sodium s u c c i n a t e pH 6.1 (1.0 ml) i n the presence of e i t h e r 2 mM iodoacetamide (o) or 2 mM i o d o a c e t i c a c i d (•). A l i q u o t s (0.1 ml) were removed from the incuba-t i o n s a t i n t e r v a l s and assayed f o r PDase I I a c t i v i t y by Method I. E x t r a p o l a t i o n o f the slower i n a c t i v a t i o n r a t e (hatched p o r t i o n ) caused fche curve to i n t e r s e c t the o r d i n a t e a t a p o i n t equal to 79% a c t i v i t y . 168 min F i g . 44: The pH dependence of i o d o a c e t a t e i n a c t i v a t i o n o f PDase I I . PDase I I (4 u n i t s ) was i n c u b a t e d w i t h 5mM i o d o a c e t i c a c i d a t 24 C i n b u f f e r mixtures (1.0ml) con-t a i n i n g 25mM sodium acetate-25mM sodium, phosphate-25mM sodium succinate-25mM sodium b o r a t e a d j u s t e d to d i f f e r e n t pH v a l u e s . A l i q u o t s (0.1ml) of the mixtures were removed a t the i n d i c a t e d times and assayed f o r r e s i d u a l P D a s t by Method I. ©, pH 9; •, pH 8; A , pH 7; O, pH 6.6; pH 6.1; A, pH 5. ;e I I 169 P r o t e c t i o n a g a i n s t the e f f e c t o f i o d o a c e t a t e Since i t was p o s s i b l e t h a t i o d o a c e t a t e was a c t i n g by a l k y l a t i n g an e s s e n t i a l a c t i v e s i t e group, experiments were c a r r i e d out to t e s t the e f f e c t i v e n e s s o f some s u b s t r a t e s i n p r o t e c t i n g the enzyme. These r e s u l t s show ( F i g . 45) t h a t dTp, ApG and pdT a l l a f f o r d e d some degree of p r o t e c t i o n whereas p o l y A was i n e f f e c t i v e . I t i s i n t e r e s t i n g t h a t w i t h the e x c e p t i o n o f pdT, the e f f e c t i v e n e s s of these compounds was i n p r o p o r t i o n s to the a f f i n i t y w i t h which they bound to the enzyme as determined e a r l i e r (Table X X I I I ) . The best p r o t e c t i o n was a f f o r d e d by dTp, a c o m p e t i t i v e i n h i b i t o r w i t h a h i g h degree o f i n t e r a c t i o n w i t h the enzyme. Comparison o f two PDase I I a c t i v i t i e s o b t ained by DNA-c e l l u l o s e chromatography I t was noted i n P a r t B t h a t chromatography o f p u r i f i e d r a t i n t e s t i n a l PDase I I on DNA-cellulose gave r i s e to two f r a c t i o n s , one of which was adsorbed to the m a t e r i a l and one which was not. In an e f f o r t to see i f these f r a c t i o n s d i f f e r e d i n s p e c i f i c i t y the enzyme a c t i v -i t i e s d e t e c t a b l e i n the o r i g i n a l p r e p a r a t i o n (see Table XXI) were measured i n the f r a c t i o n s . The r e s u l t s show t h a t o n l y PDase I I and a c i d RNAase I I a c t i v i t i e s were prese n t (Table XXVII1; t h e r e was no d e t e c t a b l e DNAase IT a c t i v i t y . I t may be noteworthy t h a t the r e l a t i v e PDase IT a c t i v i t y 170 incubation time-min F i g . 45: P r o t e c t i o n of PDase I I a g a i n s t i n a c t i v a t i o n by i o d o -a c g t i c a c i d . PDase I I (8.4 u n i t s ) was i n c u b a t e d a t 24 C i n 0.09M sodium s u c c i n a t e b u f f e r pH 6.1 (1.0ml) as d e s c r i b e d i n the legend to F i g . 43. The p r o t e c t i v e e f f e c t of Tp, pT, A p G ( a l l ImM) and p o l y A (lmg/lml) was i n v e s t i g a t e d by adding each compound t o r e a c t i o n mixtures i n the absence (o) or presence (©) of 5mM i o d o a c e t a t e . Samples (0.1ml) of these mixtures were then removed a t the i n d i c a t e d times and assayed f o r PDase I I by Method I. The smooth e x p o n e n t i a l l y -d e c r e a s i n g curve i n each diagram r e p r e s e n t s the decay of PDase I I a c t i v i t y i n the presence of 5mM i o d o a c e t i c a c i d a l o n e . 171 Table XXVII. P o l y n u c l e o t i d a s e a c t i v i t y of PDase IT f r a c t i o n s p u r i f i e d on DNA-cellulose chromatography The adsorbed and non-adsorbed f r a c t i o n s were prepared as d e s c r i b e d i n the M a t e r i a l s and Methods s e c t i o n and c o n t a i n e d 3.56 and 4.04 PDase I I u n i t s per ml r e s p e c t -i v e l y . P o r t i o n s o f these p r e p a r a t i o n s were assayed a g a i n s t dTpDNP, dTpNP, DNA and RNA as d e s c r i b e d i n the M a t e r i a l s and Methods s e c t i o n . R e l a t i v e A c t i v i t y (%) Sub s t r a t e non-adsorbed adsorbed dTpDNP 100 100 dTpNP 52 53 DNA 0 0 RNA 3.9 3.4 172 toward the s y n t h e t i c PDase s u b s t r a t e was a l s o the same i n both f r a c t i o n s (.Table XXVII I. Moreover, s i n c e b o t h f r a c t i o n s d i s p l a y e d i d e n t i c a l i n a c t i v a t i o n k i n e t i c s i n i o d o a c e t a t e s o l u t i o n s i t i s d i f f i c u l t to know what might be the b a s i s f o r the d i f f e r e n t chromatographic behaviour of these e n z y m e f f r a c t i o n s . D i f f e r e n t chromatographic forms of other n u c l e a s e s are a l s o known; f o r example the A and B forms o f p u r i f i e d p a n c r e a t i c RNAase which d i f f e r i n carbohydrate content (Reinhold e t a l . , 1968) and the A, B and C forms of p a n c r e a t i c DNAase I which are caused by d i f f e r e n c e s i n the t o t a l amide c o n c e n t r a t i o n (Laskow-s k i , 1971). Of these two, the l a t t e r i s more l i k e l y t o be r e s p o n s i b l e f o r the d i f f e r e n c e s observed w i t h PDase I I s i n c e , as p r e v i o u s l y mentioned, no carbohydrate m a t e r i a l was d e t e c t a b l e i n p u r i f i e d PDase p r e p a r a t i o n s . D i s c u s s i o n B r o a d l y speaking, the r a t i n t e s t i n a l PDase I I has been shown t o be f a t h e r s i m i l a r t o enzymes o f t h i s type fromoother sources (Hilmoe, 1960; R a z z e l l £ Khorana, 1961; F i e r s and Khorana, 1963; B e r n a r d i & B e r n a r d i , 1968; van V e n r o o i j & Poort, 1970 and Menon £ Smith, 1970). Mixtures of o l i g o n u c l e o t i d e s , d i n u c l e o t i d e s and s y n t h e t i c chromo-genic phosphodiesters were a l l h y d r o l y s e d r a p i d l y a t pH v a l u e s between 6 and 7. Since the enzyme was i n a c t i v e 173 toward or d i d not i n t e r a c t w i t h such compounds as g l y c e r y l p h o s p h o r y l c h o l i n e , NAD, b i s - ( p - n i t r o p h e n y l ) phosphate, NPpdT, ATP, a d e n y l y l (5 ,->2 ,I guanosine and 3 ' , 5 ' - c y c l i c AMP, i t i s l i k e l y t h a t a t e r m i n a l n u c l e o s i d e 3'-phosphory1 r e s i d u e i s r e q u i r e d f o r enzyme a c t i v i t y . T h e r e f o r e i t was not s u r p r i s i n g t h a t the products o f enzyme a c t i o n , namely n u c l e o s i d e 3 1-phosphates, were found t o c o m p e t i t i v e l y i n h i b i t the r e a c t i o n . The value o f the -5 i n h i b i t o r c onstant f o r dTp was 2 x 10 M, which i n d i c a t e s a h i g h degree o f a f f i n i t y f o r the enzyme. R a z z e l l & Khorana (1961) and F i e r s & Khorana (1963) r e p o r t e d t h a t dTp a l s o i n h i b i t e d the PDase I I from spleen and L a c t o b a c i l l u s a c i d o p h i l u s r e s p e c t i v e l y but the data f o r the l a t t e r case -2 show a K i va l u e of 1.5 x 10 M; no data are a v a i l a b l e f o r the spleen enzyme. The Km o f the s u b s t r a t e used i n the _5 pre s e n t experiments, dTpDNP, was found t o be about 4 x 10 M whereas i n t e r e s t i n g l y the v a l u e f o r the more commonly used p - n i t r o p h e n y l e s t e r has been shown ( F i e r s & Khorana, 1963; Flanagan, 1970; Horwitz et al_. , 1972) to be about 10 times g r e a t e r than t h i s . Attempts t o determine the i s o e l e c t r i c p o i n t o f the enzyme d i d not g i v e c o n c l u s i v e r e s u l t s . A v a l u e o f 4.1 was obtained by i s o e l e c t r i c f o c u s i n g f o r 31 hours i n a pH g r a d i e n t from 3-10. However, when a shallower g r a d i e n t was used 3 peaks of PDase were found a t pH 2.9, 4.2 and 7.8. While i t i s tempting t o suppose t h a t t h i s r e s u l t i n d i c a t e s 174 a degree of h e t e r o g e n e i t y In the PDase, i t i s a l s o p o s s i b l e t h a t s o l u b i l i t y a r t i f a c t s gave th e observed p a t t e r n s i n c e i t i s known t h a t most p r o t e i n s are l e a s t s o l u b l e a t t h e i r i s o e l e c t r i c , p o i n t s , and furthermore t h a t any p r o t e i n s so p r e c i p i t a t e d can move to other p a r t s of the i s o e l e c t r i c f o c u s i n g column as they become i n s o l u b l e (Haglund, 1971). In view o f the i n s o l u b i l i t y and aggregation problems found e a r l i e r (Parts A and B) the r e s u l t s obtained by i s o e l e c t r i c f o c u s i n g must be i n t e r p r e t e d t h e r e f o r e , w i t h c a u t i o n . H o w e v e r i j i t l i s p u z z l i n g t h a t none of the v a l u e s quoted above can r e a d i l y e x p l a i n the chromatographic behaviour o f the PDase on CMS and DEAEC (Part B). . Since the a c t i v i t y was r e a d i l y bound to CMC a t pH 5, the p r o t e i n must be s t r o n g l y c a t i o n i c at t h i s pH, whereas b i n d i n g to DEAEC showed t h a t the enzyme was weakly a n i o n i c at pH 6.5. On t h i s b a s i s the i s o e l e c t r i c p o i n t of PDase I I must l i e between pH 5 and pH 6.5. However Peterson (197 0) c a u t i o n s a g a i n s t such reasoning s i n c e he has shown t h a t f o r i o n -exchange purposes small pockets of charged groups are more important than the o v e r a l l charge on a p r o t e i n . Thus, human carbon monoxide hemoglobin ( i s o e l e c t r i c p o i n t = 6.5) can be t i g h t l y adsorbed t o CMC a t pH 7 i n 0.01M sodium phosphate, even though i t s net charge i s n e g a t i v e . A f t e r e x t e n s i v e < % p u r i f i c a t i o n o f the r a t enzyme.by chromatography on DEAEC, CMC and agarose, t h e s e prepar-a t i o n s s t i l l c o n t a i n e d a c t i v i t i e s ^ toward DNA and 'SNA. The 175 DNAase a c t i v i t y was apparently eliminated By chromatography on DNA-cellulose but the RNAase a c t i v i t y remained assoc-iated with both eluted f r a c t i o n s . I t would be inter e s t i n g to know whether the residual a c t i v i t y towards RNA was due to a persistent RNAase or the r e s u l t of an i n t r i n s i c a c t i v i t y of the PDase, since p u r i f i e d PDases from spleen (Bernardi & Bernardi, 1968) and t e s t i s (Menon & Smith, 19701 did not contain any DNAase or RNAase. Razzell (1967 L has commented on the d i f f i c u l t y of distinguishing RNAase I I a c t i v i t y from that of PDase I I . With regard to ("contaminating enzyme a c t i v i t i e s : i t i s i n t e r e s t i n g that PDase a c t i v i t y has been found i n many DNAase II preparations. Bernardi & G r i f f e (19641 o r i g i n a l l y showed that highly p u r i f i e d preparations of hog spleen DNAase II contained a weak "PDase" a c t i v i t y . The substrate used was bis-(p-nitrophenyl) phosphate but Bernardi & G r i f f e (19641 also claimed that t h e i r p u r i f i e d DNAase II was active on the p-nitrophenyl esters of thymidine deoxyguanosine and deoxycytidine 3 1-phosphates although t h i s work was never published. Thus Bernardi and co-workers believe that t h i s PDase i s an i n t r i n s i c , a l b e i t weak property of pure DNAase II and for t h i s reason they assert (Bernardi & Bernardi, 19.681 that compounds such as dTpDNP are not v a l i d substrates for PDase IT since they can be s p l i t by DNAase IT. The re s u l t s obtained i n Part A show that there is> no need to be so sweeping'; the a c t i v i t i e s of 176 DNAase IT and PDase IT toward dTpDNP can Be d i s t i n g u i s h e d 2-By c a r e f u l l y s e l e c t i n g the assay pH and By u s i n g SO^ to i n h i B i t DNAase IT. More i m p o r t a n t l y , i t has Been shown t h a t whereas the PDase a c t i v i t y o f hog spleen DNAase p r e p a r a t i o n s has d e f i e d a l l attempts t o remove i t from t h e DNAase ( S i c a r d et a l . , 1970) the same a c t i v i t y i n cow s p l e e n (Swenson & Hodes, 1969) sheep spleen ( S l o r & Hodes, 19,701 r a t l i v e r (Delaney e t aT. , 1971). and salmon t e s t i s (Yamdmoto & B i c k n e l l , 1972) can Be completely separated from the DNAase. B e r n a r d i & B e r n a r d i (1971)1 have r e t o r t e d t h a t the evidence of Swenson and Hodes (19.69.) and S l o r and Hodes (197 0) does not " c o n s t i t u t e an acceptable, evidence a g a i n s t the two a c t i v i t i e s being c a r r i e d by the same p r o t e i n molecule". D e s p i t e t h i s i t appears t h a t the "PDase" a c t i v i t y which. B e r n a r d i & G r i f f e (1964) claimed t o be an i n t r i n s i c p r o p e r t y of hog spleen DNAase I I i s i n f a c t a t e n a c i o u s contaminant ( S l o r , 197 0). Supportive evidence f o r t h i s can be seen i n the r e s u l t s o f Oshima & P r i c e (1973) who found t h a t low c o n c e n t r a t i o n s of i o d o a c e t a t e completely i n h i B i t hog spleen DNAase without a f f e c t i n g the a s s o c i a t e d PDase. Thus i t appears t h a t s p e c i e s and organ d i f f e r e n c e s can a f f e c t the contamination of some nu c l e a s e s by a s s o c i a t e d enzymes:. T h i s should be kept i n mind i n view; o f the presence of RNAase (and perhaps DNAase I i n p u r i f i e d r a t i n t e s t i n a l PDase IT p r e p a r a t i o n whereas such, a c t i v i t i e s : a r e absent 177 from other PDase I I p r e p a r a t i o n s CBernardi & B e r n a r d i , 19-68;' Menon & Smith-, 1-9-7 0.1. A d e t a i l e d examination of the h y d r o l y s i s products r e l e a s e d By i n t e s t i n a l PDase I I from DNA and RNA would Be of i n t e r e s t , But time d i d not permit such s t u d i e s t o Be made. F i n a l l y , i t should Be p o i n t e d out t h a t the PDase a c t i v i t y associatedC.with hog s p l e e n DNAase I I is- c l e a r l y not PDase I I f o r the f o l l o w i n g reasons: Cal the s u B s t r a t e used t o estimate the PDase a c t i v i t y was B i s - C p - n i t r o p h e n y l ) phosphate which i s a v e r y poor s u B s t r a t e f o r PDase I I CBernardi & B e r n a r d i , 1968; see a l s o TaBle X X I I ) / CB) Swenson & Hodes C19691 showed t h a t B i s - ( p - n i t r o p h e n y l ) phosphate was hy d r o l y s e d much more r a p i d l y than was dTpNP By the PDase; Cc). the mol e c u l a r weight o f the enzyme must Be c l o s e t o 38,000, the va l u e o h t a i n e d By B e r n a r d i e t a l . (1965) f o r DNAase s i n c e Both the DNAase and PDase co-sedimented i n a sucrose g r a d i e n t , whereas PDase I I from d i f f e r e n t sources has a mol e c u l a r weight of 100,000 (Menon & Smith, 1969) or g r e a t e r (van V e n r o o i j & Poort, 1970 and see F i g s 17 and 35); (d) Oshima & P r i c e (1973) showed t h a t the DNAase a s s o c i a t e d PDase a c t i v i t y was u n a f f e c t e d By i o d o a c e t a t e whereas r e s u l t s o f the present work i n d i c a t e t h a t PDase I I was q u i c k l y i n a c t i v a t e d i n the presence of t h i s a l k y l a t i n g agent. The r e s u l t s o b tained w i t h a numBer o f enzyme i n h i b i t o r s ' m e r i t d i s c u s s i o n . S i n c e o r g a n i c m e r c u r i a l 178 reagents (PCMB, PCMPS). and NEM i n h i b i t e d PDase i t might be expected t h a t s u l p h y d r y l groups are i n v o l v e d i n the c a t a l y t i c process to some ext e n t . T h i s c o n c l u s i o n i s ++ supported by the s t r o n g i n h i b i t i o n observed w i t h Cu (Table XXV ) s i n c e copper i s a common poiso n e r of s u l p h y d r y l enzymes ( V a l l e e & Riordan, 1969). A number of noteworthy f i n d i n g s were ob t a i n e d w i t h the a l k y l a t i n g reagent i o d o a c e t i c a c i d . In the presence of 5mM i o d o a c e t a t e PDase IT was r a p i d l y i n a c t -i v a t e d . F i f t y percent of the a c t i v i t y was d e s t r o y e d i n 20-30min at 24°C and at pH. v a l u e s between 5 and 6.1, c o n d i t i o n s under which the enzyme i s normally completely s t a b l e . A r e l a t e d a l k y l a t i n g agent, iodoacetamide was t o t a l l y without e f f e c t . The r a t e of the a l k y l a t i o n r e a c t i o n was slower at h i g h e r pH v a l u e s , or when a s u b s t r a t e o r a c o m p e t i t i v e i n h i b i t o r was p r e s e n t . The l a t t e r r e s u l t i s v e r y good evidence t h a t the r e s i d u e being a l k y l a t e d i s a t the a c t i v e c e n t r e . Since the r e s u l t s o b tained with, the other i n h i b i t o r s had i m p l i e d the involvement o f SH- groups i n the a c t i o n of the enzyme i t was i n i t i a l l y supposed t h a t an e s s e n t i a l s u l p h y d r y l group was being a l k y l a t e d . However t h i s view had t o be m o d i f i e d because iodoacetamide, a more r e a c t i v e t h i o l a l k y l a t i n g reagent (Dixon & Webb, 19.64L was without e f f e c t . Besides- c y s t e i n e , i o d o a c e t a t e can a l k y l a t e the e-amino groups of l y s i n e residues-, the i m i d a z o l e groups of h i s t i d i n e residues-, t h e p h e n o l i c h y d r o x y l s of t y r o s i n e 179 r e s i d u e s , the t h i o e t h e r sulphur atoms o f methionine r e s i d u e s and the £5 and y c a r b o x y l groups o f a s p a r t i c ' and glutamic a c i d s r e s p e c t i v e l y . However a l k y l a t i o n o f l y s i n e r e s i d u e s i s most r a p i d a t hig h e r pH v a l u e s (Gundlach e t a l . , 1959a) and the i n a c t i v a t i o n o f PDase I I of n e u t r a l or s l i g h t l y a c i d i c pH v a l u e s was probably too r a p i d to support the p o s s i b i l i t y t h a t a methionine was being a l k y l a t e d (Gundlach e t a l . , 1959b). Of the remaining p o s s i b i l i t i e s the most l i k e l y i s r e a c t i o n a t a h i s t i d i n e i f o n l y because a l k y l a t i o n of an e s s e n t i a l h i s t i d i n e has been shown t o i n a c t i v a t e RNAase A (Gundlach et a l . , 1959a; Stark e t a l . , 1961; F r u c h t e r & C r e s t f i e l d , 1965), DNAase I ( P r i c e et a l . , 1969). and DNAase I I COshima & P r i c e , 1973);. Carboxy-m e t h y l a t i o n o f an e s s e n t i a l g l utamic a c i d r e s i d u e i n RNAase by i o d o a c e t a t e has been accomplished by Takahashi e t a l . (1967).. In each o f these cases i o d o -acetamide, a n e u t r a l a l k y l a t i n g agent, was i n e f f e c t i v e as i t was i n the present case a g a i n s t PDase I I . Since the r e a c t i v i t i e s o f i o d o a c e t a t e and iodoacetamide toward a - N - a c e t y l -L - h i s t i d i n e are q u i t e ^ s i m i l a r (Stark e t a l . , 1961), the s t r i k i n g d i f f e r e n c e s cannot be a s c r i b e d t o i n t r i n s i c d i f f e r e n c e s i n the r e a c t i v i t y o f these two reagents towards i m i d a z o l e , Rather i t appears t h a t t h e r e i s a p o s i t i v e l y charged group i n or c l o s e t o the a c t i v e s i t e o f a l l o f these enzymes which a t t r a c t s t h e i o d o a c e t a t e ^nion-tbi^a,??;d t h e 180 a c t i v e s i t e where i t carBoxymethylates the a p p r o p r i a t e r e s i d u e . The pH dependence of the i n a c t i v a t i o n o f PDase TT By i o d o a c e t a t e supports the i d e a t h a t such a p o s i t i v e l y charged group c o u l d Be t h e imidazolium p o r t i o n o f a h i s t i d i n e r e s i d u e s i n c e i n c r e a s i n g the pH above 7 lowered the a l k y l a t i o n r a t e markedly. Such a mechanism has a l r e a d y been demonstrated f o r RNAase (Stark et a l . 1961). where one i m i d a z o l e a t t r a c t s the n e g a t i v e l y charged a l k y l a t i n g agent w h i l e the other p r o v i d e s the e l e c t r o n s t o d i s p l a c e i o d i n e from i t and t h e r e f o r e becomes carboxymethylated. Knowledge of which r e s i d u e i s m o d i f i e d by i o d o a c e t a t e i n PDase I I must await r e a c t i o n o f the enzyme w i t h r a d i o a c t i v e i o d o a c e t a t e and c h a r a c t e r i z a t i o n o f the i n a c t i v e p r o t e i n by amino a c i d a n a l y s i s . I t i s i n t r i g u i n g t h a t enzymes w i t h such d i f f e r e n t s p e c i f i c i t i e s as RNAase, DNAase, I, DNAase I I , RNAase 1^, and PDase I I should e x h i b i t such s t r i k i n g s i m i l a r i t i e s at t h e i r active, s i t e r e g i o n s , as i n d i c a t e d by t h e i r s i m i l a r r e a c t i o n w i t h i o d o a c e t i c a c i d . 181 When a l l t h a t s t o r y ' s f i n i s h e d , what's the news? In l u c k or out the t o i l has l e f t i t s mark: -- Yeats (1933) 182 BIBLIOGRAPHY A l l f r e y , V. & M i r k s y , A. E. (1952) J . Gen. P h y s i o l . 36, 227-241 Ames, B. N. (1966) M e t h . E n z y m o 1 . 8 , 115-118 A n d r e w s , P. (1965) B i o c h e m . J . 96, 595-606 A n f i n s e n , C. B., C u a t r e c a s a s , P. & T a n i u e h i , H. (1971) i n The  Enzymes ( B o y e r , P. D. e d . ) , v o l . 4, 3 r d e d n . , pp. 177-204 A c a d e m i c P r e s s , New Y o r k a n d L o n d o n B a r n a r d , E. A. (1969a) Ann. Rev. B i o c h e m . 38, 677-732 B a r n a r d , E. A. (1969b) N a t u r e (London) 2 2 1 , 340-344 B a r r e l l , B. G. & C l a r k , B. F. C. (1974) Handbook o f N u c l e i c A c i d  S e q u e n c e s , J o y n s o n - B r u v v e r s L t d . , O x f o r d B a u t z , E. K. F. & Dunn, J . J . (1971) i n P r o c e d u r e s i n N u c l e i c A c i d  R e s e a r c h ( C a n t o n i , G. L. & D a v i e s , D~. R~. e d s . ) , v o l . 2~r pp. 743-74 7, H a r p e r and Row, New Y o r k and L o n d o n B e a u f a y , H. (1972) i n L y s o s o m e s , A L a b o r a t o r y Handbook ( D i n g l e , J . T . e d . ) , pp. 1-45, N o r t h - H o l l a n d , L o n d o n a nd Am s t e r d a m B e a u f a y , H., J a c q u e s , P., B a u d h u i n , P., S e l l i n g e r , O. Z., B e r t h e t , J . &d£eDuve, C. (1964) B i o c h e m . J . 92, 184-205 B e h n k e , O. & Moe, H. (1964) J . C e l l B i o l . 22, 633-652 B e r n a r d i , A. & B e r n a r d i , G. (1968) B i o c h e m . B i o p h y s . A c t a 1 5 5 , 360-370 B e r n a r d i , A. & B e r n a r d i , G. (1971) i n The Enzymes ( B o y e r , P. D. e d . ) , v o l . 4, 3 r d e d n . , pp. 329-33 6 , A c a d e m i c P r e s s , New Y o r k a n d L o n d o n B e r n a r d i , G. (1971) i n The Enzymes ( B o y e r , P. D. e d . ) , v o l . 4, 3 r d e d n . , pp. 2 7 1 - 2 8 7 , A c a d e m i c P r e s s , New Y o r k a n d L o n d o n B e r n a r d i , G. & B e r n a r d i , A. (1966) i n P r o c e d u r e s i n N u c l e i c A c i d  R e s e a r c h ( C a n t o n i , G. L. & Davies"^ DT R ! e d s . ) , v o l . T~, pp. 1 4 4 - 1 5 3 , H a r p e r a nd Row, New Y o r k a nd L o n d o n B e r n a r d i , G. & G r i f f e , M. (1964) B i o c h e m i s t r y 3, 1419-1426 B e r n a r d i , G., A p p e l l a , E. & Z i t o , R. (1965) B i o c h e m i s t r y 4, 1725-1729 B r o w n , D. M., H e p p e l , L. A. & H i l m o e , R. J . (1954) J . Chem. S o c . 40-46 183 B u l l , H. B. (1971) An Introduction: to P h y s i c a l B i o c h e m i s t r y , 2nd edn., F. A. Davis Co., P h i l a d e l p h i a Burdon, R. H. (1971) Progr. N u c l e i c A c i d Res. Mol. B i o l . 11, 33-79 C a r r a r a , M. & B e r n a r d i , G. (1968) B i o c h e m i s t r y 7, 1121-1132 Chrambach, A. (1966) A n a l . Biochem. 15, 544-548 C l a r k , B. & Porteous, J . W. (1965) Biochem. J . 96, 539-551 Connock, M. & Pover, W. (1970) Histochem. J . 2, 371-380 Cunningham, L. & Laskowski, M. (1953) Biocheirm. Biophys. A c t a 11, 590-591 D a l z i e l , K. (1973) i n Rate C o n t r o l o f B i o l o g i c a l Processes (Davies, D. D. ed.), pp. 21-48, Cambridge U n i v e r s i t y Press Davis, B. J . (1964) Ann. N. Y. Acad. S c i . 121, 404-427 \ deDDu\ve,CC. (1963) i n Ciba Found. Symp. Lysosomes (deReuck, A. V. S. & Cameron, M. P. ed s . ) , pp. 1-35, C h u r c h i l l , London deDDuive,DC, (19 65) Harvey L e c t . 59, 49-87 depDuve,CC.& Wattiaux, R. (19 66) Ann. Rev. P h y s i o l . 28, 435-492 deDDu«ve.,CC,. Pressman, B. C. , G i a n e t t o , R. , Wattiaux, R. & Appelmans, F. (1955) Biochem. J . 60, 604-617 Dixon, M. (1953) Biochem. J . 55, 170-171 Dixon, M. & Webb, E. C. (19 64) Enzymes, 2nd edn., Longmans, London Drummond, G. I. & Yamamoto, M. (1971) i n The Enzymes (Boyer, P. D. ed.), v o l . 4, 3rd edn., pp. 355-371, Academic P r e s s , New York and London Dulaney, J . T., Touster, 0. & Coleman, V. (1972) J . B i o l . Chem. 247, 1424-1432 E r e c i n s k a , M., Sierakowska, H. & Shugar, D. (1969) Eur. J . Biochem. 11, 465-471 Evans, E. M., Wrigglesworth, J . M., Bu r d e t t , K. & Pover, W. F. R. (1971) J . C e l l B i o l . 51, 452-464 F i e r s , W. & Khorana, H. G. (1963) J . B i o l . Chem. 238, 2780-2788 Flanagan, P. R. (1970) M.Sc. T h e s i s , U n i v e r s i t y of B r i t i s h Columbia Flanagan, P. S. Zbarsky,7,kS. (1972) Proc. Can. Fed. B i o l . Soc. 15, 517 184 F l a n a g a n , P. &' Z b a r s k y , &. (1973) P r o c . C an. F e d . B i o l . S o c . 1 6 , 314 F l a n a g a n , P. Z b a r s k y ( 1 9 7 4 ) B i o c h e m . J . i n p r e s s F r u c h t e r , R. G. & C r e s t f i e l d , A. M. (1965) J . B i o l . Chem. 240, 38«Z5-3882 F u t a i , M. & M i z u n o , D. (1967) J . B i o l . Chem. 2 4 2 , 5301-5307 G r e s h a m , P. A. & P o v e r , W. F. R. (1967) I n t . J . R a d i a t . B i o l . 13 275-278 G u n d l a c h , H. G., S t e i n , W. H. & M o o r e , S. (1959a) J . B i o l . Chem. 234, 1754-1760 G u n d l a c h , H. G., S t e i n , W. H. & M o o r e , S. (1959b))) J . B i o l . Chem. 234, 1761-1764 H a g l u n d , H. (1971) M e t h . B i o c h e m . A n a l . 1 9 , 1-104 H a y a i s h i , 0. (1955) M e t h . E n z y m o l . 1, 668-670 H e n n i n g , R., P l a t t n e r , H. & S t o f f e l , W. (1973) B i o c h e m . B i o p h y s . A c t a 3 3 0, 61-75 H e p p e l , L. A. (1955) M e t h . E n z y m o l . 2, 570-576 H e p p e l , L. A. (1967) M e t h . E n z y m o l . 1 2 , 316-317 H e p p e l , L. A. & H i l m o e , R. J . (1955) M e t h . E n z y m o l . 2, 565-570 H i l m o e , R. J . (1960) J . B i o l . Chem. 2 3 5 , 2117-2121 Hje : r t e e 2 n , S. (1962) B i o c h i m . B i o p h y s . A c t a 6 2 , 445-449 H o f f m a n , L. G. & M c G i v e r n , P. W. (1969) J . C h r o m a t o g . 40, 53-61 H o l l a n d e r , V. P. (1971) i n The Enzymes ( B o y e r , P. D. ed.) v o l . 4, 3 r d e d n . , p p . 449 - 4 9 8 , A c a d e m i c P r e s s , New Y o r k a n d L o n d o n H o l l e y , R. W., A p g a r , J . , E v e r e t t , G. A. M a d i s o n , J . T., M a r q u i s s e , M., M e r i l l , S. H., P e n w i c k , J . R. & Z a m i r , A. (1965) S c i e n c e 1 47, 1462-1465 H o r w i t z , J . P., E a s w a r r a n , C. V. & W o l f , P. L. (1972) B i o c h i m . B i o p h y s . A c t a 276, 206-214 H s u , L. & T a p p e l , A. L. (1964) J . C e l l B i o l . 2 3 , 233-240 H u b s c h e r , G. & W e s t , G. R. (1965) N a t u r e (London) 2 05, 799-800 H u b s c h e r , G., W e s t , G. R. & B r i n d l e y , D. N. (1965) B i o c h e m . J . 97, 629-642 185 Hynie, I . & Zbarsky, S. H. (1970a) Can. J . Biochem. 48, 1141-1150 Hynie, I. & Zbarsky, S. H. (1970b) Can. J . Biochem. 48, 1151-1159 Iwanoff, L. (1903) Z. P h y s i o l . Chem. 38, 31-43 Joss e , J . , K a i s e r , A. D. & Kornberg, A. (1961) J . B i o l . Chem. 236, 864-875 Kapitany, R. A. & Zebrowski, E. J . (1973) A n a l . Biochem. 56 361-369 Karnovsky, M. J . (1965) J . C e l l B i o l . 27, 137A-138A Khorana, H. G. (1961) i n The Enzymes (Boyer, P. D., Lardy, H. & Myrback, K. ed s . ) , v o l . 5, 2nd edn., pp. 79-94, Academic P r e s s , New York and London K i r b y , A. J . & V a r v o g l i s , A. G. (1968) J . Chem. Soc. (B) 135-141 K l e i n , W. & Thannhauser, S. J . (1935) Z. P h y s i o l . Chem. 231, 96-103 Kognig, H. (1969) i n Lysosomes i n B i o l o g y and Pathology (Dingle, J.T. ed.) , v o l . 2, pp. 111-162, North-Holland, London and Amsterdam Kornberg, A. (1969) Science 163, 1410-1418 Krebs, H. A. & Veech, R. L. (1969) i n The Energy L e v e l and M e t a b o l i c  C o n t r o l i n Mi t o c h o n d r i a (Papa,5S., Tager, J . M., Q u a g l i a r i e l l o , E. & S l a t e r , E. C. ed s . ) , B a r i : A d r i a t i c a E d i t r i c e Kumamoto, J . , Raison, J . K. & Lyons, J . M. (1971) J . Theoret. B i o l . 31, 47-51 K u n i t z , M. (1950) J . Gen. P h y s i o l . 33, 349-362 Laskowski, M. (1961) i n The Enzymes (Boyer, P. D., Lardy, H. & Myrback, K. e d s . ) , v o l . 5, 2nd edn., pp. 123-147, Academic Press, New York and London Laskowski, M. (1971) i n The Enzymes (Boyer, P. D. ed.), v o l . 4, 3rd edn., pp. 289-311, Academic Press, New York and London Laskowski, M. & F i l i p o w i c z , B. (1958) B u l l . Soc. Chim. B i o l . 40, 1865-1873 Laurent, T. C. & Kiilander, J . (1964) J . Chroma tog. 14, 317-330 Leblond, C. P. & Stevens, C. E. (1948) Anat. Rec. 100, 357-371 Lehman, I. R. (1960) J . B i o l . Chem. 235, 1479-1487 Lehman, I. R. (1963) Progr. N u c l e i c A c i d Res. 2, 83-123 186 Lehman, I. R. (1967) Ann. Rev. Biochem. 36, 645-668 Lehman, I . R. (1971) i n The Enzymes (Boyer, P. D. ed.), v o l . 4, 3rd edn., pp. 251-270, Academic P r e s s , New York and London Le i g h t o n , F., Poole, B., Beaufay, H., Baudhuin, P., C o f f e y , J . W., Fowler, S. & deDuve, C. (1968) J . C e l l B i o l . 37, 482-513 Levene, P. A. & Bass, L. W. (1931) N u c l e i c A c i d s , The Chemical C a t a l o g Co., New York Levene, P. A. & Medigreceanu, F. (1911a) J . B i o l . Chem. 9, 65-83 Levene, P. A. & Medigreceanu, F. (1911b) J . . B i o l . Chem. 9, 375-387 Levene, P. A. & Medigreceanu, F. (1911c) J . B i o l . Chem. 9, 389-402 Lineweaver, H. & Burk, D. (1934) J . Amer. Chem. Soc. 56, 658-666 Litman, R. M. (1968) J . B i o l . Chem. 243, 6222-6233 Lowry, D. H., Rco'sceb^rough, N. J . , F a r r , A. L. & R a n d a l l , R. J . (1951) J . B i o l . Chem. 193, 265-275 L u f t , J . H. (1961) J . Biophys. Biochem. C y t o l . 9, 409-414 Markham, R. & Smith, J . D. (1952) Biochem. J . 52, 565-571 Maver, M. E. & Greco, A. E. (1949) J . B i o l . Chem. 181, 853-860 Menon, K. M. J . & Smith, M. (1970) Bio c h e m i s t r y 9, 1584-1592 Meselson, M. , Yuan, R. & Heywood, J . (1972) Ann. Rev. Biochem. 41, 447-466 Mizuno, D. & Anraku, Y. (1967) Japan J . Med. S c l . B i o l . 20, 127-149 Moe, H., Rostgaard, J . & Behnke, 0. (1965) J . U l t r a s t r u c t . Res. 12, 396-403 : Moore, S. & S t e i n , W. H. (1973) S c i e n c e 180, 458-464 Oshima, R. G. & P r i c e , P. A. (1973) J . B i o l . Chem. 248, 7522-7526 Pennington, R. J . (1961) Biochem. J . 80, 649-654 Peterson, E. A. (1970) i n Laboratory Techniques i n Bi o c h e m i s t r y and  Mol e c u l a r B i o l o g y (Work, T. S. & Work, E. eds.) v o l . 2~, pp. 223-400, North-Holland, Amsterdam and London Porath, J . (1962) Nature (London) 196, 47-48 187 Porteous, J . W. (1972) i n S u b c e l l u l a r Components/ P r e p a r a t i o n and  F r a c t i o n a t i o n ( B i r n i e , G. D. ed.), 2nd edn., pp. 157-183, Butterworths, London Porteous, J . W. & C l a r k , B. (19 65) Biochem. J . 96, 159-171 P r i c e , P. A., Moore, S. & S t e i n , W. H. (1969) J . B i o l . Chem. 244, 924-928 Rahman, Y. E. (1967) Biochim. Biophys. A c t a 146, 477-483 Raison, J . K. (1973) B i o e n e r g e t i c s 4, 285-309 Rauenbusch, E. C. & Altman, K. L. (1960) Proc. Soc. Exp. B i o l . Med. 104, 385-388 R a z z e l l , W. E. (1961) J . B i o l . Chem. 236, 3028-3030 R a z z e l l , W. E. (1967) E x p e r i e n t i a 23, 321-325 R a z z e l l , W. E. (1968) Can. J . Biochem. 46, 1-7 R a z z e l l , W. E. & Khorana, H. G. (1961) J . B i o l . Chem. 236, 1144-1149 Reid, E. (1972) i n S u b c e l l u l a r Components, P r e p a r a t i o n and F r a c t i o n a t l o n ( B i r n i e , G. DT ed.), 2nd edn., pp. 93-118, Butterworths, London Reinhold, V., Dunne, F. T., Wriston, J . C., Schwartz, M., Sardhu, L. & H i r s , C. H. W. (1968) J . B i o l . Chem. 243, 6482-6494 Robinson, G. B. (1963) Biochem. J . 88, 162-168 Schachman, H. K. (1959) U l t r a c e n t r i f u g a t i o n i n Bi o c h e m i s t r y , Academic P r e s s , New York and London Schmidt, G. & Laskowski, M. (1961) i n The Enzymes (Boyer, P. D., Lardy, H. & Myrback, K. ed s . ) , v o l . 5, 2nd edn., pp. 3-35 Academic P r e s s , New York and London Schneider, W. C. (1957) Meth. Enzymol. 3, 680-684 S.garamella, V. & Khorana, H. G., (1972) J . Mol. B i o l . 72, 427-444 S i c a r d , P. J . , Obrenovitch, A. & Aubel-Sadron, G. (1970) FEBS L e t t . 12, 41-44 Sierakowska, H., Szemplinska, H. & Shugar, D. (1963) A c t a Biochim. Pate. 10, 399-415 S l o r , H. (1970) Biochem. Biophys. Res. Comm. 38, 1084-1090 188 S l o r , H. & Hodes, M. E. (1970) A r c h . B l o c h e m . B i o p h y s . 138, 172-173 Stark, G. R., S t e i n , W. H. & Moore, S. (1961) J . B i o l . Chem. 236, 436-442 — S t r a u s , W. (1956) J . B i o p h y s . B i o c h e m . C y t o l . 2, 513-521 Swenson, M. K. & H o d e s , M. E. (1969) J . B i o l . Chem. 244, 1803-1807 Takahashi, K., S t e i n , W. H. & Moore, S. (1967) J . B i o l . Chem. 242, 4682-4690 - T r i e r , J . S. (1968) i n The Handbook of P h y s i o l o g y (Code, C. F. ed.) S e c t i o n 6, v o l . I I I ^ pp. 1125-1175, American P h y s i o l o g i c a l S o c i e t y , Washington, D. C. T u r n b u l l , J . M. & N e i l , M. W. (1969) Biochem. J . I l l , 503-507 Uchida, T. & Egami, F. (1971) i n The Enzymes (Boyer, P. D. ed.), v o l . 4, 3rd edn., p p . 205-250, Academic P r e s s , New York and London Uzawa, S. (1932) J . B i o c h e m . (Tokyo) 15, 19-28 V a l l e e , B. L. & R i o r d a n , J . F. (1969) A n n . Rev. B i o c h e m . 38, 733-794 v a n D y c k , J . M. & W a t t i a u x , R. (1968) E u r . J . B i o c h e m . 7, 15-20 v a n V e n r o o i j , W. J . W. & P o o r t , C. (1970) E u r . J . B i o c h e m . 13, 391-397 V e s t e r b e r g , 0. & S v e n s s o n , H. (19 66) A c t a Chem. S c a n d . 20, 820-834 v o n T i g e r s t r o m , R. & S m i t h , M. (1969) B i o c h e m i s t r y , 8, 3067-3070 W a t t i a u x , R., W i b o , M. & B a u d h i u n , P. (1963) i n C i b a F o u n d . Symp. L y s o s o m e s (de R e u c k , A. V. S. & Cameron, M. P. e d s . ) , pp. 17 6-200, C h u r c h i l l , L o n d o n W o l f , P. L., H o r w i t z , J . P., F r e i s l e r , J . V., V a z q u e z , J . & Von d e r M u e h l l , E. (1968) B i o c h i m . B i o p h y s . A c t a , 1 5 9 , 212-214 W r i g g l e s w o r t h , J . M. & P o v e r , W. F. R. (1966) L i f e S c i . 5, 1365-1371 Yamamoto, M. & B i c k n e l l , L. H. (1972) A r c h . B i o c h e m . B i o p h y s . 151, 261-269 Y e a t s , W. B. (1933) f r o m "The C h o i c e " i n The W i n d i n g S t a i r a n d O t h e r  Poems, M a c m i l l a n a n d Co. L t d . , L o n d o n Z a r u b a , F., K a r a s e k , M. A. & F a r b e r , E. M. (1967) J . I n v e s t . D e r m a t o l . 49, 537-543 

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