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Purification and kinetics of xanthine dehydrogenase from two wild-type isoalleles of Drosophila melanogaster Edwards, Thomas Charles Robert 1977

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P u r i f i c a t i o n and Kinetics of Xanthine Dehydrogenase from Two Wild-Type I s o a l l e l e s of Vn.o&oph<lla MzlanoQO&tzn. by Thomas Charles Robert Edwards B.Sc.(Agr), University of B r i t i s h Columbia, 1974 A Thesis Submitted i n P a r t i a l F u l f i l l m e n t of the Requirements for the Degree of Master of Science i n The Faculty of Graduate Studies Department of Biochemistry We accept this thesis as conforming to the required standard The University of B r i t i s h Columbia May, 1977 Thomas Charles Robert Edwards In presenting th is thes is in p a r t i a l fu l f i lment of the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, J agree that the L ibrary sha l l make it f ree ly ava i lab le for reference and study. I fur ther agree that permission for extensive copying of th is thes is for scho la r ly purposes may be granted by the Head of my Department or by his representat ives . It is understood that copying or pub l ica t ion of th is thes is for f i n a n c i a l gain sha l l not be allowed without my wri t ten permission. Department of B i o c h e m i s t r y The Univers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 7 TABLE OF CONTENTS i Page INTRODUCTION 1 A. G e n e t i c B a c k g r o u n d 1 B. X a n t h i n e D e h y d r o g e n a s e s 2 C. K i n e t i c s a n d I n h i b i t i o n o f XDH 4 D. G e n e r a l L i g a n d s f o r A f f i n i t y C h r o m a t o g r a p h y 5 E. I m m u n o a f f i n i t y C h r o m a t o g r a p h y 6 F. The P r e s e n t I n v e s t i g a t i o n 7 MATERIALS AND METHODS 9 M a t e r i a l s 9 A b b r e v i a t i o n s 10 METHODS 11 A. B u f f e r s 11 B. A s s a y o f Enzyme A c t i v i t y 11 C. P r e p a r a t i o n and P u r i f i c a t i o n o f XDH 13 D. P r o d u c t i o n o f A n t i b o d i e s 15 E. I m m u n o d i f f u s i o n 16 F. I m m u n o a f f i n i t y C h r o m a t o g r a p h y 17 G. S y n t h e s i s o f 9 - ( p - a m i n o e t h o x y p h e n y l ) g u a n i n e 18 H. P r e p a r a t i o n o f B l u e D e x t r a n - S e p h a r o s e CL-4B 24 I . P r o t e i n D e t e r m i n a t i o n 24 RESULTS AND DISCUSSION 25 A. I m m u n o a f f i n i t y C h r o m a t o g r a p h y 25 B. I m m u n o d i f f u s i o n 33 C. K i n e t i c s 37 D. A f f i n i t y C h r o m a t o g r a p h y 54 CONCLUSION 56 LIST OF TABLES + 4 K values for XDH from fiu 1409H and m J h.y+^1409H with xanthine, hypoxanthine and NAD+ i i i L I S T OF FIGURES F i g u r e s Page 1 O u t l i n e o f t h e s y n t h e s i s o f 9 - ( p -a m i n o e t h o x y p h e n y l ) g u a n i n e 21 2 E f f e c t o f NH4SCN c o n c e n t r a t i o n on XDH a c t i v i t y 27 3 P u r i f i c a t i o n o f ry+4i4 09 XDH on a n t i b o d y - S e p h a r o s e 30 4 S c a n o f SDS-PO , 5% p o l y a c r y l a m i d e g e l o f ry+4i409H XDH a f t e r p u r i f i -c a t i o n on a n t i b o d y - S e p h a r o s e 32 5 I m m u n o d i f f u s i o n o f XDH e x t r a c t s i n 1% A g a r o s e 35 6 Enzyme k i n e t i c s o f XDH f r o m vy+4i409E and ry + Hi409N. D o u b l e r e c i p r o c a l p l o t s o f 1/v v e r s u s 1 / [ S u b s t r a t e ] . 39 7 P i n g - p o n g r e a c t i o n mechanism f o r XDH 41 8 I n h i b i t i o n o f ry+4i409H XDH by 9 - ( p -a m i n o e t h o x y p h e n y l ) g u a n i n e 46 9 P r o p o s e d i n h i b i t i o n scheme f o r t h e i n h i b i t i o n o f XDH b y 9 - ( p - a m i n o e t h o x y -p h e n y l ) g u a n i n e 49 10 R e p l o t o f s l o p e 1/v f r o m 1/v v e r s u s 1 / [ h y p o x a n t h i n e ] p l o t v e r s u s c o n c e n t r a -t i o n o f 9 - ( p - a m i n o e t h o x y p h e n y l ) g u a n i n e 51 11 R e p l o t o f a p p a r e n t 1 / V m a x f r o m 1/v v e r s u s 1 / [ h y p o x a n t h i n e ] p l o t v e r s u s c o n c e n t r a t i o n o f 9 - ( p - a m i n o e t h o x y p h e n y l ) g u a n i n e 53 ACKNOWLEDGEMENTS I would l i k e to express my thanks to my super-v i s o r , Dr. Peter Candido f o r his excellent advice, assistance and encouragement throughout the course of my studies. Many thanks go to Flor a Grabowska for r a i s i n g the f l i e s and for her technical assistance. To my wife Barbara, I give my warmest appreciation for her patience, encouragement and moral support. I t i s a pleasure to acknowledge the personal support of the Medical Research Council of Canada i n the form of a studentship from 19 74 to 19 77. ABSTRACT X a n t h i n e d e h y d r o g e n a s e (XDH) f r o m two s t r a i n s o f Vno&ophila. m<LlcLnoQ.CL&t<Lfi d i f f e r i n g i n t h e 1409 s i t e was s t u d i e d . The 1409U a l l e l e , i s t h e w i l d - t y p e and t h e . 1 4 0 9 H v a r i a n t h a s b e e n p r e v i o u s l y shown t o g i v e g r e a t e r XDH a c t i v i t y p e r f l y . The two s t r a i n s , iy+^<L409U and ny*^ X.40 9W p r o d u c e XDH m o l e c u l e s o f t h e same e l e c t r o p h o r e t i c m o b i l i t y . + 4 . I n t h i s s t u d y , XDH f r o m h.y <L409H was p u r i f i e d b y ammonium s u l f a t e f r a c t i o n a t i o n , h e a t i n g a t 6 0 ° f o r 5 m i n u t e s , D E A E - c e l l u l o s e c h r o m a t o g r a p h y , and p r e p a r a t i v e e l e c t r o -p h o r e s i s , and u s e d t o r a i s e a n t i b o d i e s a g a i n s t D r o s o p h i l a XDH i n r a b b i t s . The a n t i s e r u m p r o d u c e d gave two p r e c i p i t i n l i n e s when d i f f u s e d a g a i n s t c r u d e f l y e x t r a c t , o n l y one o f w h i c h s t a i n e d f o r XDH a c t i v i t y . D i f f u s i o n a g a i n s t e x t r a c t s o f Ky-n u l l o m u t a n t s gave: one p r e c i p i t i n l i n e w h i c h d i d n o t s t a i n f o r XDH a c t i v i t y , was i d e n t i c a l t o t h e n o n s t a i n i n g l i n e o f w i l d - t y p e e x t r a c t s , and gave a r e a c t i o n o f p a r t i a l i d e n t i t y w i t h t h e X D H - a c t i v e p r e c i p i t i n l i n e . C o u p l i n g t h e a n t i b o d i e s t o S e p h a r o s e CL-4B gave an e f f e c t i v e m a t e r i a l f o r i m m u n o - a f f i n i t y c h r o m a t o g r a p h y . XDH f r o m h e a t - t r e a t e d e x t r a c t s was a d s o r b e d t o t h e a n t i b o d y -S e p h a r o s e , t h e column was washed w i t h 1M N a C l , and t h e XDH was e l u t e d w i t h 2.4M NH 4SCN. As h i g h c o n c e n t r a t i o n s o f NH^ SCN c a u s e d r a p i d i n a c t i v a t i o n o f XDH, t h e enzyme was i m m e d i a t e l y d e s a l t e d b y p a s s a g e t h r o u g h Sephadex G-25. A f t e r i m m u n o a f f i n i t y c h r o m a t o g r a p h y , XDH gave o n l y one p r e c i p i t i n l i n e on immuno-v i d i f f u s i o n . P u r i f i e d XDH e x h i b i t e d two h i g h m o l e c u l a r w e i g h t p o l y p e p t i d e b a n d s on e l e c t r o p h o r e s i s i n s o d i u m d o d e c y l s u l p h a t e . The k i n e t i c s o f XDH p u r i f i e d by i m m u n o a f f i n i t y i t a t o g r a p h + 4 c h r o m a t o g r a y w e r e s t u d i e d . XDH f r o m b o t h h.y+^1409H and fiy Z409H e x h i b i t e d o r d e r e d b i n d i n g f o r s u b s t r a t e and NAD i n a p i n g - p o n g r e a c t i o n mechanism. The enzyme f r o m Jiy+^<L409N -5 e x h i b i t e d a K f o r x a n t h i n e o f 2.4 x 10 M, a K f o r h y p o x -ia m -5 + -5 a n t h i n e o f 1.6 x 10 M and a K f o r NAD o f 4.0 x 10 M. m + 4 Enzyme f r o m ny Z4 09H e x h i b i t e d s i m i l a r k i n e t i c c o n s t a n t s . T h i s r e s u l t p r o v i d e s f u r t h e r e v i d e n c e t h a t t h e Z409H a l l e l e r e s u l t s i n a h i g h e r s t e a d y s t a t e number o f XDH m o l e c u l e s p e r f l y , and d o e s n o t a l t e r t h e k i n e t i c p r o p e r t i e s o f t h e enzyme. An i n h i b i t o r o f x a n t h i n e d e h y d r o g e n a s e s , 9 - ( p -a m i n o e t h o x y p h e n y l ) g u a n i n e was s y n t h e s i z e d and t h e i n h i b i t i o n o f XDH d e t e r m i n e d . The r e s u l t s o b t a i n e d i n d i c a t e d t h a t 9 - ( p -a m i n o e t h o x y p h e n y l ) g u a n i n e i s a d e ad-end i n h i b i t o r , and b i n d s t o b o t h o x i d i z e d and r e d u c e d XDH w i t h a R. o f 4.55 x 10 6 M -7 and 5.7 x 10 M r e s p e c t i v e l y . A t t e m p t s a t a f f i n i t y c h r o m a t o g r a p h y o f XDH w i t h i m m o b i l i z e d 9-(p-aminoethoxyphenyl) g u a n i n e , w h i c h i s known t o b i n d b o v i n e x a n t h i n e o x i d a s e , p r o v e d u n s u c c e s s f u l . The l a c k o f b i n d i n g o f XDH may be due t o s t e r i c f a c t o r s . Two n u c l e o t i d e co-enzyme a n a l o g u e s , B l u e D e x t r a n and i m m o b i l i z e d NADv'are known t o be e f f e c t i v e l i g a n d s f o r enzymes p o s s e s s i n g t h e d i n u c l e o t i d e f o l d . XDH d i d n o t b i n d t o t h e s e a n a l o g u e s , i n d i c a t i n g t h a t XDH may n o t p o s s e s s t h e d i n u c l e o t i d e f o l d . 1 INTRODUCTION A . G e n e t i c B a c k g r o u n d I n Vno&ophUba. melanogaAteA x a n t h i n e d e h y d r o g e n a s e (XDH) m o l e c u l e i s a homodimer, p o s s e s s i n g two c o p i e s o f t h e p o l y p e p t i d e t h a t i s c o d e d f o r by t h e s t r u c t u r a l e l e m e n t o f t h e r o s y [ty] l o c u s ( 1 - 1 1 ) . The ny l o c u s i s a g e n e t i c u n i t l o c a t e d on chromosome 3, and h a s a map l e n g t h o f 5.0 x 10 map u n i t s ( 2 ) . I t was o r i g i n a l l y d e f i n e d by a s e t o f r e c e s s i v e b r o w n i s h e y e c o l o r m u t a n t s . The fiy gene has b e e n t h e s u b j e c t o f i n t e n s i v e f i n e s t r u c t u r e a n a l y s i s by C h o v n i c k and c o - w o r k e r s w h i c h has g i v e n a l i n e a r map o f n u l l m u t a n t s , i n t e r a l l e l i c c o m p l e m e n t i n g m u t a n t s , d e t e r m i n a n t s o f t h e g e n e t i c b a s i s o f v a r i a t i o n i n e l e c t r o p h o r e t i c m o b i l i t y o f XDH i n w i l d - t y p e i s o a l l e l e s , and p u r i n e s e n s i t i v e " l e a k y " s t r u c t u r a l m u t a n t s (2, 3, 6, 12, 1 3 ) . A p u t a t i v e c o n t r o l v a r i a n t has b e e n d e s c r i b e d (3) t h a t l i e s midway between t h e l e f t m o s t unambiguous s t r u c t u r a l 606 v a r i a n t (<ty an i n t e r a l l e l i c complementer) , and t h e n e x t g e n e t i c e l e m e n t £ ( 3 ) S / 2 . T h i s v a r i a n t , d e s i g n a t e d 14 09, i s a c i s - a c t i n g r e g u l a t o r o f t h e fiy s t r u c t u r a l e l e m e n t . Two a l l e l e s have b e e n described,X . 4 0 9 N w h i c h g i v e s n o r m a l o r w i l d -t y p e amounts o f XDH a c t i v i t y p e r f l y , and 1409H w h i c h g i v e s i n c r e a s e d amounts o f XDH a c t i v i t y p e r f l y . The i n c r e a s e d a c t i v i t y was shown by i m m u n o t i t r a t i o n t o be due t o a g r e a t e r number o f XDH m o l e c u l e s p e r f l y . T h i s i s n o t l i k e l y due t o 2. a g r e a t e r enzyme s t a b i l i t y i n JL40W: f l i e s as XDH f r o m b o t h a l l e l e s h ad t h e same t h e r m o l a b i l i t y a t 6 0 ° . P r e l i m i n a r y e x a m i n a t i o n o f t h e k i n e t i c s o f XDH f r o m t h e two a l l e l e s showed no d i f f e r e n c e s i n a f f i n i t y f o r h y p o x a n t h i n e . B. X a n t h i n e D e h y d r o g e n a s e s X a n t h i n e d e h y d r o g e n a s e s f r o m Mlcn.0 coccu.& lactyiZyt-ica6 ( 1 4 ) , Colia.i> b u t t e r f l i e s ( 1 5 ) , Vno&ophlla me,lanoga6tzi ( 1 ) , r a t ( 1 6 ) , c h i c k e n ( 1 7 ) , t u r k e y ( 1 8 ) , and b o v i n e m i l k (19) a r e a l l m o l y b d o f l a v o p r o t e i n s c o n t a i n i n g 2 m o l e s o f FAD, 2 m o l e s o f molybdenum a n d 8 mo l e s o f non-heme i r o n p e r mole o f enzyme, w i t h m o l e c u l a r w e i g h t s o f 250,000 t o 300,000 and s u b u n i t m o l e c u l a r w e i g h t s o f 125,000 t o 150,000. The enzymes f r o m d i f f e r e n t s o u r c e s do d i f f e r i n e l e c t r o n a c c e p t o r s p e c i f i c i t y . The enzymes f r o m b o v i n e m i l k and UttcAo coccuA lact-LZyt-LcuA a r e o x i d a s e s as t h e y a r e (^-depend-e n t and c a n n o t u t i l i s e NAD + as an e l e c t r o n a c c e p t o r (14, 1 9 ) . R a t l i v e r x a n t h i n e d e h y d r o g e n a s e has been r e p o r t e d t o be a NAD*-dependent d e h y d r o g e n a s e i n c r u d e e x t r a c t s , w i t h i n c r e a s -i n g o x i d a s e a c t i v i t y o n p u r i f i c a t i o n (16, 20, 21, 22) . The c o n v e r s i o n f r o m d e h y d r o g e n a s e a c t i v i t y t o o x i d a s e a c t i v i t y c a n be a c h i e v e d i n s e v e r a l ways, n o t a b l y by h e a t t r e a t m e n t and l i m i t e d p r o t e o l y s i s w i t h t r y p s i n . The u n t r e a t e d d e h y d r o g e n a s e and h e a t - t r e a t e d o x i d a s e enzymes had s u b u n i t m o l e c u l a r w e i g h t s o f 150,000 w h i l e t h e t r y p s i n ^ t r e a t e d enzyme h a d a s l i g h t l y s m a l l e r s u b u n i t o f 130,000 m o l e c u l a r w e i g h t . 3. The enzymes f r o m c h i c k e n and t u r k e y l i v e r a r e a l s o N A D + - d e p e n d e n t d e h y d r o g e n a s e s w i t h some o x i d a s e a c t i v i t y (17, 18, 23, 24) . Collai> and Vn.o&ophlla x a n t h i n e d e h y d r o -g e n a s e s a r e NAD - d e p e n d e n t w i t h no r e p o r t e d o x i d a s e a c t i v i t i e s . A l l o f t h e enzymes a r e a b l e t o u s e a r t i f i c i a l e l e c t r o n a c c e p t o r s t o v a r y i n g e x t e n t s . Some o f t h e e l e c t r o n a c c e p t o r s t h a t have b e e n u s e d a r e 2 , 6 - d i c h l o r o i n d o p h e n o l ( D C I P ) , p h e n a z i n e m e t h o s u l f a t e , m e t h y l e n e b l u e , t r i n i t r o -b e n z e n e s u l f o n a t e , a n d f e r r i c y a n i d e . DCIP i s commonly u s e d a s an e l e c t r o n a c c e p t o r i n enzyme a s s a y s . M i l k x a n t h i n e o x i d a s e h as two b i n d i n g s i t e s f o r DCIP w i t h a K,. = 1.3 x 1 0 6 M ( 2 5 ) . The dye i n t e r a c t s w i t h t h e d i s s enzyme a t , o r n e a r t h e s u b s t r a t e b i n d i n g s i t e and i s a n o n -c o m p e t i t i v e , m i x e d - t y p e i n h i b i t o r w i t h K^=2.8 x 10 M. F o r m a t i o n o f t h e DCIP enzyme complex i s n o t r e q u i r e d f o r -7 e l e c t r o n t r a n s f e r as DCIP has a K = 1.5 x 10 M a s an m e l e c t r o n a c c e p t o r . C h a o t r o p i c a g e n t s s u c h as KI and KSCN have b e e n shown t o d i s s o c i a t e t h e FAD c o f a c t o r f r o m c h i c k e n (26, 27) and t u r k e y (27) enzymes and b o v i n e m i l k x a n t h i n e o x i d a s e ( 2 6 ) . Vn.o&opklla XDH i s a l s o i n a c t i v a t e d by KI (1) , p r e s u m a b l y b y t h e same mechanism. R e d u c t i o n o f XDH b y s u b s t r a t e o r NADH, i n t h e a b s e n c e o f e l e c t r o n a c c e p t o r s , p r i o r t o e x p o s u r e t o KI r e n d e r s t h e FAD c o f a c t o r more r e a d i l y d i s s o c i a b l e . As t h e x a n t h i n e o x i d o - r e d u c t a s e s have a l a b i l e p e r s u l f i d e g r o u p a t t h e i r a c t i v e s i t e s , p r e r e d u c t i o n w i t h s u b s t r a t e w i l l o n l y a f f e c t f u n c t i o n a l enzyme w h i l e p r e r e d u c t i o n w i t h NADH w i l l 4. a f f e c t both functional and desulfo enzyme (27-30). Using t h i s c r i t e r i o n , XDH p u r i f i e d by c l a s s i c a l prodedures commonly contains 20% to 50% of non-functional enzyme (28-30). C. Kinetics and I n h i b i t i o n of XDH The k i n e t i c properties of xanthine oxido-reductases are very s i m i l a r . Chicken (17), turkey (18) and bovine milk (19) enzymes e x h i b i t ping pong reaction mechanisms. This involves two separate half reactions with the ordered binding of two substrates, with the addition of the f i r s t substrate being at thermodynamic equilibrium. Previous investigations of the k i n e t i c s of VKo&opk-lla. XDH (31, 32) , have not reported a ping pong reaction mechanism. Baker and co-workers synthesized numerous derivatives of 9-phenylguanine which were i n h i b i t o r s of milk xanthine oxidase (33-36). One compound 9-(p-aminoethoxyphenyl) guanine was a strong inhibitor- and an excellent ligand for the a f f i n i t y chromatography of r a t l i v e r xanthine dehydrogenase and guanine deaminase (35). They reported a 230-fold p u r i -f i c a t i o n of xanthine dehydrogenase a c t i v i t y with 90% recovery. Fogle and Bieber (37) have reported a 1250-fold p u r i f i c a t i o n of guanine aminohydrolase from rabbit l i v e r on a column of 9-(p-aminoethoxyphenyl) guanine-Sepharose. A l l o p u r i n o l (4-hydroxypyrazolo [3,4-d]pyrimidine) i s another very strong i n h i b i t o r of xanthine oxido-reductases. Massey zt al. (38) and Spector and Johns (39) showed that a l l o p u r i n o l i s a substrate of milk xanthine oxidase and the t-product allo-xanthine (4,6-dihydroxypyrazolo [3,4-d] pyrimidine) 5. b i n d s t o t h e reduced enzyme w i t h a K,. = 5.4 x 10 ^ M ( 3 9 ) . d i s s The enzyme bound molybdenum i s t r a p p e d i n t h e reduced Mb I V s t a t e and i s unable t o pass t h e r e d u c i n g e q u i v a l e n t s t h r o u g h t h e i n t e r n a l e l e c t i o n t r a n s p o r t c h a i n t o 0^ ( 3 8 ) . The e x t r e m e l y low K ^ s g f o r t h e a l l o - x a n t h i n e - e n z y m e complex r e s u l t s i n a t i t r a t i n g o r st o i c h i o m e t r i c ; i n h i b i t i o n . D. G e n e r a l L i g a n d s f o r A f f i n i t y Chromatography Many p r o t e i n s w h i c h b i n d n u c l e o t i d e s o r n u c l e o t i d e co-enzymes p o s s e s s a s u p e r - s e c o n d a r y s t r u c t u r e c a l l e d t he d i n u c l e o t i d e f o l d . T h i s i s a s t r u c t u r e o f about 120 amino a c i d s a r r a n g e d i n ayfi - s h e e t c o r e composed o f f i v e o r s i x p a r a l l e l s t r a n d s c o n n e c t e d by C ( - h e l i c a l i n t r a s t r a n d l o o p s l o c a t e d above and below the jS-sheet (40,41) . I t i s known t o form t h e N A D + - b i n d i n g s i t e o f l a c t a t e ( 4 2 ) , m a l a t e ( 4 3 ) , and g l y c e r a l d e h y d e - p h o s p h a t e (44) dehydrogenases, t h e A T P - b i n d i n g s i t e o f p h o s p h o g l y c e r a t e k i n a s e (45) and t o be p r e s e n t i n t h e s t r u c t u r e s o f a l c o h o l dehydrogenase ( 4 6 ) , a d e n y l a t e k i n a s e (47,48) and phospho^-glycerate mutase ( 4 9 ) . C i b a c r o n b l u e F3GA, t h e chromophore o f B l u e D e x t r a n , has been shown t o be an an analogue o f NAD + and t o i n t e r a c t w i t h p r o t e i n s c o n t a i n i n g t h e d i n u c l e o t i d e f o l d ( 50-52). I t i s a c o m p e t i t i v e i n h i b i t o r f o r t h e s u b s t r a t e NADH o f M^ and H 4 — 7 —6 l a c t a t e dehydrogenase w i t h K. o f 1.3 x 10 M and 2.1 x 10 M r e s p e c t i v e l y ( 5 2 ) . B l u e Dextran-Sepharose i s an e f f e c t i v e a f f i n i t y chromatography s u p p o r t f o r t h e enzymes l i s t e d above w h i c h c o n t a i n t h e d i n u c l e o t i d e f o l d . I n a d d i t i o n r i b o n u c l e a s e , and f e r r o c y t o c h r o m e C w h i c h do not c o n t a i n t h e f o l d s t r u c t u r e 6. b i n d t o B l u e D e x t x a n - S e p h a r o s e , as do p h o s p h o g l u c o n a t e d e -h y d r o g e n a s e , p h o s p h o f r u c t o k i n a s e , f r u c t o s e d i p h o s p h a t a s e , and DNA p o l y m e r a s e where t h e p r e s e n c e o f t h e f o l d i s unknown. A l l o f t h e enzymes e x c e p t . r ; i b o n u c l e a s e b i n d t o B l u e D e x t r a n -S e p h a r o s e i n 0.01M T r i s - H C l pH 7.5 b u f f e r and c a n be e l u t e d w i t h a low c o n c e n t r a t i o n o f s u b s t r a t e (1-10 mM), o r h i g h i o n i c s t r e n g t h (1M N a c i ) . I m m o b i l i z e d NAD and AMP have a l s o b e en r e p o r t e d t o be e f f e c t i v e g e n e r a l l i g a n d s f o r s e v e r a l N A D + - s p e c i f i c d e -h y d r o g e n a s e s (53, 5 4 ) . Ox h e a r t l a c t a t e and r a b b i t g l y c e r a l -d e h y d e - 3 - p h o s p h a t e d e h y d r o g e n a s e s r e a d i l y b i n d t o b o t h NAD-S e p h a r o s e and AMP-Sepharose ( 5 3 ) . T hey a r e r e a d i l y e l u t e d f r o m NAD-Sepharose w i t h 0.15 mM NADH o r 0.15 mM NAD r e s p e c t i v e l y . -4 The K m ( N A D H ) f o r o x h e a r t l a c t a t e d e h y d r o g e n a s e i s 1 x 10 M ( 5 5 ) , and ( N A D ) f ° r r a b b i t g l y c e r a l d e h y d e - 3 - p h o s p h a t e d e --5 h y d r o g e n a s e i s 5 x 10 M ( 5 6 ) . Qn AMP-Sepharose, n e i t h e r enzyme i s e l u t e d b y s a l t c o n c e n t r a t i o n s o f up t o 0.5 M KC1. B i n d i n g t o t h e i m m o b i l i z e d NAD and AMP i s n o t common t o a l l d e h y d r o g e n a s e s . G l y c e r a l d e h y d e - 3 - p h o s p h a t e d e h y d r o g e n a s e f r o m Bac<LlluA i>tZdHoth<i>vmoph.X,LvJ> b i n d s t o NAD-Sepharose b u t n o t t o AMP-Sepharose. A l c o h o l d e h y d r o g e n a s e f r o m t h e same s o u r c e b i n d s t o AMP-Sepharose b u t n o t t o NAD-SepharQ.se ( 5 4 ) . E. I m m u n o a f f i n i t y C h r o m a t o g r a p h y B i o - s p e c i f i c c h r o m a t o g r a p h y u s i n g t h e s p e c i f i c i t y o f a n t i b o d i e s h as b e e n w i d e l y e x p l o i t e d f o r immunoassays and f o r t h e p u r i f i c a t i o n o f b i o l o g i c a l m o l e c u l e s . 7. Antibodies coupled to CNBr-activated Sepharoses re t a i n t h e i r s p e c i f i c i t y , although there may be a loss i n a f f i n i t y and number of combining s i t e s (57). To elute the bound antigen from an antibody column, strong d i s s o c i a t i n g agents such as chaotropic s a l t s , urea, extremes of pH or ^'hapten (1, 58-60) are used. The chaotropic thiocyanate ion has been reported to be a very e f f e c t i v e agent for the d i s s o c i a t i o n of antigen-antibody complexes i n immunoadsorption chromatography (59,60). Anderson zt al. (59) found that 3M NH^ SCN completely eluted bound c a l f serum proteins without reducing the binding capacity of the column. Decreasing the NH4SCN concentration below 3M gave incomplete e l u t i o n . Other chaotropic s a l t s that have been used include MgCl 2 (61) and KI (1). Treatment of immobilized antibodies with the reagent 2-hydroxy-5-nitrobenzyl bromide greatly f a c i l i t a t e s the e l u t i o n of bound antigens (58). This treatment does not greatly reduce the binding capacity of the immunoadsorbent, but rather reduces the a f f i n i t y of the a n t i -bodies for antigen. The major i n t e r f e r i n g e f f e c t i n immunoadsorption i s non-biospecific binding, e s p e c i a l l y when crude preparations of antibody or antigen are employed. Pre-elution of the column material with 1M s a l t i s e f f e c t i v e i n e l u t i n g non-b i o s p e c i f i c a l l y bound material (62) . Antibodies can be raised against proteins homogenized i n polyacrylamide gels a f t e r electrophoresis under eith e r non-denaturing (3, 63), or denaturing (6 4) conditions. This 8 . p e r m i t s t h e p u r i f i c a t i o n o f p r o t e i n s f o r i m m u n i z a t i o n b y g e l e l e c t r o p h o r e s i s w i t h o u t h a v i n g t o e l u t e t h e p r o t e i n f r o m t h e g e l . The i n e r t p o l y a c r y l a m i d e g e l p r o b a b l y a c t s as an e f f e c t i v e a d j u v a n t , b u t i s h o m o g e n i z e d w i t h Freund's c o m p l e t e a d j u v a n t as. w e l l . A n t i s e r u m t o VnobophLla XDH has b e e n p r e p a r e d by A n d r e s (1) u s i n g enzyme p u r i f i e d by t h e p r o c e d u r e o f S e y b o l d ( 6 5 ) , and by C h o v n i c k zt al.(3) a g a i n s t XDH f r o m s e v e r a l w i l d - t y p e and m utant s t r a i n s p o o l e d t o g e t h e r and f r a c t i o n a t e d by pH5 p r e c i p i t a t i o n , h e a t i n g a t 5 6 ° , a m m o n i u m s u l f a t e , S e phadex G-150 and p o l y a c r y l a m i d e s l a b g e l e l e c t r o p h o r e s i s . F - The P r e s e n t I n v e s t i g a t i o n T h i s i n v e s t i g a t i o n e x t e n d s t h e k i n e t i c a n a l y s i s o f D r o s o p h i l a XDH i n i t i a t e d by C h o v n i c k zt al. (3) . A d e t a i l e d e x a m i n a t i o n o f t h e k i n e t i c p a r a m e t e r s o f homogeneous XDH f r o m Hy Z40 9H and ny Z409H d i d n ° t r e v e a l any d i f f e r e n c e s i n a f f i n i t y f o r s u b s t r a t e s . The i n h i b i t i o n o f XDH by 9 - ( p -a m i n o e t h o x y p h e n y l ) , g u a n i n e was i n v e s t i g a t e d and shown t o be due t o d e a d - e n d inM^bii'tiion o f t h e o x i d i x e d and r e d u c e d forms o f t h e enzyme. A t t e m p t s were made t o p u r i f y XDH by a f f i n i t y c h r o m a t o g r a p h y on 9 - ( p - a m i n o e t h o x y p h e n y l ) g u a n i n e - S e p h a r o s e 6B, B l u e D e x t r a n - S e p h a r o s e CL-4B and NAD-Agarb.se w i t h o u t s u c c e s s . 9. MATERIALS AND METHODS MATERIALS The f o l l o w i n g m a t e r i a l s were u s e d : 2-amino-4^ c h l o r o - 6 - p y r i m i d i n o l m o n o h y d r a t e ( A l d r i c h C h e m i c a l Co.) ; a g a r o s e ( B i o - R a d L a b o r a t o r i e s ) ; Freund,'scomplete a d j u v a n t ( D i f c o L a b o r a t o r i e s ) ; a c r y l a m i d e , N , N ' - m e t h y l e n e b i s a c r y l a m i d e c y a n o g e n b r o m i d e , s o d i u m p - n i t r o p h e n o x i d e (Eastman O r g a n i c C h e m i c a l s ) ; s o d i u m d o d e c y l s u l f a t e (B.D.H.); a c e t i c a n h y d r i d e , d i m e t h y l f o r m a m i d e , d i m e t h y l s u l f o x i d e , formamide, f o r m i c a c i d , g l y c e r o l , g l y c i n e , N o r i t A, p o t a s s i u m i o d i d e , s e a s a n d , s o d i u m a z i d e , and z i n c d u s t ( F i s h e r S c i e n t i f i c C o . ) ; ammonium t h i o c y a n a t e , 1 , 2 - d i b r o m o e t h a n e , h e x a m e t h y l e n e t e t r a m i n e ( M a l l i n c k r o d t ) ; Whatman DE-52 (Mand e l S c i e n t i f i c ) ; 2 , 6 - d i c h -l o r o i n d o p h e n o l , f$ - m e r c a p t o e t h a n o l , 5 - m e t h y l p h e n a z i n i u m m e t h y l s u l f a t e , (Matheson, Coleman and B e l l ) ; S e p hadex G-25 medium, S e p h a r o s e 6B, S e p h a r o s e CL-4B ( P h a r m a c i a F i n e C h e m i c a l s ) ; B l u e D e x t r a n 2000, c r y s t a l l i n e B o v i n e serum a l b u m i n (BSA), h y p o -x a n t h i n e , x a n t h i n e , NAD + ( g r a d e I I I and V ) , n i t r o b l u e t e t r a -z o l i u m , p h e n y l m e t h y l s u l f o n y l f l u o r i d e (PMSF), 2-amino-4-hydroxy-p t e r i n , s o d i u m m e r t h i o l a t e , T r i z m a b a s e (Sigma C h e m i c a l C o . ) ; B o v i n e m i l k x a n t h i n e o x i d a s e ( W o r t h i n g t o n B i o c h e m i c a l C o r p . ) ; 6 + N -NAD - A g a r o s e (P-L B i o c h e m i c a l s ) . P l a t i n u m o x i d e was a g i f t f r o m Dr. G. T e n e r . L a b o r a t o r y s t o c k s o f Vn.o&ophlZ.a. me.la.no<ga&to.it were f r o m Dr. A. C h o v n i c k , U n i v e r s i t y o f C o n n e c t i c u t , S t o r r s , C o n n e c t i c u t . A l l o f t h e s t o c k s u s e d h a v e b e e n d e s c r i b e d p r e v -i o u s l y (2,3) . T h r e e l a r g e , m a l e New Z e a l a n d w h i t e r a b b i t s were 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 , 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 . L I S T OF ABBREVIATIONS AEPG 9 - ( p - a m i n o e t h o x y p h e n y l ) g u a n i n e BSA B o v i n e serum a l b u m i n DCIP 2 , 6 - d i c h l o r o i n d o p h e n o l Hx h y p o x a n t h i n e NAD + g - n i c o t i n a m i d e a d e n i n e d i n u c l e o t i d e PMS 5 - m e t h y l p h e n a z i n i u m m e t h y l s u l f a t e PMSF p h e n y l m e t h y l s u l f o n y l f l u o r i d e SDS s o d i u m d o d e c y l s u l f a t e XO X a n t h i n e O x i d a s e XDH X a n t h i n e d e h y d r o g e n a s e METHODS A. B u f f e r s 1. B u f f e r A: 0.1 M T r i s - H C l pH 8.0, 4 mM p -m e r c a p t o e t h a n o l . 2. B u f f e r B: 0.01 M T r i s - H C l pH 8.0, 1.3 mM jS -m e r c a p t o e t h a n o l . 3. B u f f e r P: 0.1 M T r i s - H C l pH 8.0, 1.3 mM / S -m e r c a p t o e t h a n o l , 1 mM e t h y l e n e d i a m i n e t e -t r a c e t i c a c i d , 0.2 M N a C l , and 0.01% sod i u m m e r t h i o l a t e . B. A s s a y o f Enzyme A c t i v i t y 1. 2,6-Dichloroindophenol A s s a y : Enz;yme a c t i v i t y was a s s a y e d by f o l l o w i n g t h e r e d u c t i o n o f 2 , 6 - d i c h l o r o i n d o p h e n o l by s u b s t r a t e i n the Vpresence o f XDH. The a s s a y mix c o n t a i n s O.1 M T r i s - H C l pH 8.0, 1 mg/ml NAD +, 1 mg/ml B o v i n e Serum A l b u m i n and 60 ^ g / m l d i c h l o r o i n d o p h e n o l . To a s s a y XDH a c t i v i t y , 0.05 m l o f enzyme e x t r a c t , and 0.4 ml o f a s s a y m i x were i n c u b a t e d i n a c u v e t t e . When endogenous a c t i v i t y was e x h a u s t e d 0.05 ml o f s u b s t r a t e s o l u t i o n was added and t h e d e c r e a s e i n A g 0 g r e c o r d e d u s i n g a G i l f o r d r e c o r d i n g s p e c t r o -p h o t o m e t e r . T h i s a s s a y h a s t h e d i s a d v a n t a g e t h a t a ny r e d u c i n g a g e n t s , s u c h a s ^ - m e r c a p t o e t h a n o l p r e s e n t i n t h e enzyme e x t r a c t w i l l a l s o r e d u c e t h e d y e . 2. >NAD A s s a y : Enzyme a c t i v i t y was a s s a y e d b y f o l l o w i n g t h e f o r m a t i o n o f NADH f r o m NAD + a t 340 nm. F o r t h e r o u t i n e a s s a y o f col u m n f r a c t i o n s , a 0.05 ml a l i q u o t o f enzyme was added t o 0.45 ml o f a s s a y mix composed o f 0.1 M T r i s - H C l pH 8.0, 1 mg/ml B o v i n e Serum Albumin, 1 mM x a n t h i n e and 0.75 mM NAD +, and t h e r a t e o f r e d u c t i o n o f NAD + r e c o r d e d . F o r t h e d e t e r m i n a t i o n o f k i n e t i c c o n s t a n t s , s t o c k s o l u t i o n s o f a s s a y b u f f e r (0.1M T r i s - H C l pH 8.0, 1 mg/ml B o v i n e Serum A l b u m i n ) , s u b s t r a t e and NAD + were p r e p a r e d . A r e a c t i o n mix o f 0.35 ml a s s a y b u f f e r , 0.05 ml NAD + s o l u t i o n and 0.05 m l o f s u b s t r a t e was i n c u b a t e d a t 2 8 ° f o r f i v e m i n u t e s , t h e n 0.05 m l o f enzyme was added and t h e r a t e o f r e d u c t i o n o f NAD + f o l l o w e d a t 340 nm. 3. F l u o r i m e t r i c A s s a y : The f l u o r i m e t r i c a s s a y o f XDH u s e d was t h a t o f C h o v n i c k tt al. (66) . The o x i d a t i o n o f 2 - a m i n o - 4 - h y d r o x y p t e r i d i n e t o i s o x a n t h o p t e r i n i n t h e p r e s e n c e o f XDH and NAD +, was mea s u r e d by t h e i n c r e a s e i n f l u o r e s c e n c e a t 412 nm when i r r a d i a t e d , ! a t 335 nm. The f l u o r i m e t e r was s e t t o z e r o f l u o r e s c e n c e w i t h t h e r e a c t i o n mix, w h i c h was 0.1 M T r i s - H C l pH 8.0, 0.01 M a m i n o h y d r o x y p t e r i n , 1 mg/ml NAD +, 1 mg/ml BSA, and f u l l s c a l e d e f l e c t i o n o f t h e r e c o r d e r was a d j u s t e d t o 117 mV o u t p u t . A l l measurements were p e r -f o r m e d on a B a i r d - A t o m i c F l u o r e s c e n c e s p e c t r o p h o t o m e t e r , M o d e l SF-1 e q u i p p e d w i t h a d i g i t a l v o l t m e t e r and a H o n e y w e l l E l e c t r o n i k 194 c h a r t r e c o r d e r . 13. C. P r e p a r a t i o n and P u r i f i c a t i o n o f XDH 1. E x t r a c t i o n and F r a c t i o n a t i o n E x t r a c t i o n and f r a c t i o n a t i o n was performed e s s e n t i a l l y as d e s c r i b e d by S e y b o l d ( 6 5 ) . F o r p r e p a r a t i o n s of up t o 80 grams o f f l i e s , t h e f l i e s were ground by hand i n a c h i l l e d m o r t a r i n 4 volumes o f B u f f e r A, 1% N o r i t A and 0.5 grams o f sand p e r gram o f f l i e s . P r e p a r a t i o n s g r e a t e r ' t h a n 80 grams were homo-g e n i z e d i n a S o r v a l l Omni-mix i n 3 volumes o f B u f f e r A, and 1% o f N o r i t A. The homogenate was c e n t r i f u g e d a t 10,000 xg f o r 15 mi n u t e s and t h e s u p e r n a t a n t f i l t e r e d t h r o u g h g l a s s w o o l . A s u f f i c i e n t amount o f 100 mM PMSF i n n - b u t a n o l was added t o make t h e s u p e r n a t a n t 1 0 0 ^ M PMSF. The s u p e r n a t a n t was b r o u g h t t o 30% s a t u r a t i o n w i t h c o l d s a t u r a t e d (NH^JjSO^ i n 0.1 M T r i s - H C l pH 8.0, s t i r r e d f o r 30 minutes and t h e p r e c i p i t a t e removed by c e n t r i f u g a t i o n a t 10,000 xg f o r 20 min u t e s . The s u p e r n a t a n t was t h e n made 60% s a t u r a t e d i n (NH^) 2 SO^, s t i r r e d f o r 30 minutes and t h e p r e c i p i t a t e c o l l e c -t e d by c e n t r i f u g a t i o n a t 10,000 xg f o r 20 m i n u t e s . The p e l l e t was suspended i n B u f f e r B and heated i n g l a s s t u b e s a t 60° f o r 5 m i n u t e s , c h i l l e d i m m e d i a t e l y i n an i c e b a t h , and c e n t r i f u g e d a t 12,000 xg f o r 10 m i n u t e s . The s u p e r n a t a n t was d e s a l t e d by d i a l y s i s a g a i n s t B u f f e r B, th e n e i t h e r a p p l i e d d i r e c t l y t o a column o f D E A E - c e l l u l o s e o r s t o r e d f r o z e n a t -20° u n t i l used. 14 . 2. D E A E - C e l l u l o s e C h r o m a t o g r a p h y H e a t - t r e a t e d e x t r a c t s p r e p a r e d as d e s c r i b e d were f u r t h e r f r a c t i o n a t e d by s t e p w i s e e l u t i o n f r o m D E A E - c e l l u l o s e . H e a t - t r e a t e d e x t r a c t s were a p p l i e d t o D E A E - c e l l u l o s e e q u i l i b r a t e d w i t h B u f f e r B and t h e col u m n washed w i t h 0.1 M N a C l i n B u f f e r B u n t i l t h e a b s o r b a n c e a t 280 nm was l e s s t h a n 0.05. The XDH was t h e n e l u t e d f r o m t h e c o l u m n w i t h 0.2M N a C l i n B u f f e r B. The f r a c t i o n s c o n t a i n i n g XDH a c t i v i t y were p o o l e d , made 60% s a t u r a t e d i n ( N H ^ ) 2 SO^ and t h e p r e c i p i t a t e c o l l e c t e d a f t e r c e n t r i f u g a t i o n a t 20,000 xg f o r 15 m i n u t e s . The p e l l e t was d i s s o l v e d i n B u f f e r B and d e s a l t e d by d i a l y s i s a g a i n s t B u f f e r B. 3. P r e p a r a t i v e E l e c t r o p h o r e s i s The T r i s - g l y c i n e b u f f e r s y s t e m w h i c h r u n s a t pH 8.9 ( D a v i s (67) ) , w i t h o u t a s t a c k i n g g e l , was u s e d . S t o c k s o l u t i o n s were made up as f o l l o w s : A, 48 ml 1 N H C 1 , 36.3 grams T r i s b a s e , 0.45 m l N, N, N 1 , 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 and w a t e r t o a volume o f 100 m l ; B, 45 grams a c r y -l a m i d e , 1.2 grams m e t h y l e n e b i s a c r y l a m i d e and w a t e r t o 100 m l ; C, 14 mg ammonium p e r s u l f a t e i n 10 ml w a t e r ( f r e s h l y made). F o r 5% g e l s , t h e s o l u t i o n s were m i x e d i n t h e p r o p o r t i o n s 1A/0.87 B/2.11 w a t e r / 4 C . F o r 7.5% g e l s t h e p r o p o r t i o n s were 1A/1.3 B/1.7 w a t e r / 4 C . S t o c k r e s e r v o i r b u f f e r was 12 grams T r i s b a s e , 57.6 grams g l y c i n e and w a t e r t o 1 l i t e r , d i l u t e d 1/10 t o u s e . Up t o 1 ml o f p a r t i a l l y p u r i f i e d XDH f r o m a D E A E - c e l l u l o s e c o l u m n ( e q u i v a l e n t t o 10 grams o f f l i e s ) was a p p l i e d t o a 1.0 cm x 10 cm 5% g e l and e l e c t r o p h o r e s e d a t 3 t o 4 mA/gel u n t i l t h e b r o m o p h e n o l b l u e t r a c k i n g dye r e a c h e d t h e end o f t h e g e l ( a b o u t 4 h o u r s ) . The g e l was t h e n removed and s t a i n e d f o r XDH a c t i v i t y a s d e s c r i b e d u n t i l t h e t e t r a -z o l i u m b a n d was j u s t v i s i b l e . The s e c t i o n o f g e l c o n t a i n i n g t h e XDH a c t i v i t y was c u t o u t and h o m o g e n i z e d i n 0.01 M T r i s -H C l pH 8.0, made 10% i n g l y c e r o l and 0.002% b r o m o p h e n o l b l u e , and r e a p p l i e d t o a 7.5% g e l o f t h e same d i m e n s i o n s . The m a t e r i a l f r o m as many as s i x 5% g e l s c o u l d be a p p l i e d t o one 7.5% g e l . A f t e r e l e c t r o p h o r e s i s f o r up t o 7 h o u r s , t h e g e l was l i g h t l y s t a i n e d f o r XDH a c t i v i t y arid t h e band e x c i s e d . G e l s l i c e s c o n t a i n i n g XDH were s t o r e d f r o z e n a t - 2 0 ° u n t i l u s e d . On a 7.5% a n a l y t i c a l g e l C o o m a s s i e b l u e s t a i n i n g r e v e a l e d a s i n g l e p r o t e i n band w h i c h was c o i n c i d e n t w i t h XDH a c t i v i t y . D. P r o d u c t i o n o f A n t i b o d i e s A n t i s e r u m t o D r o s o p h i l a XDH was r a i s e d i n t h r e e l a r g e m a l e r a b b i t s . The r a b b i t s were i n j e c t e d i n t r a p e r i t o n e -a l l y on a 1, 3, 7, 10, 17, 23 day s c h e d u l e . F o r t h e f i r s t two i n j e c t i o n s o f e a c h r a b b i t , 7.5% g e l bands c o n t a i n i n g XDH e q u i v a l e n t t o 50 grams o f f l i e s were h o m o g e n i z e d w i t h 0.5 m l 0.9% N a C l , t h e n e m u l s i f i e d w i t h an e q u a l volume' o f F r e u n d ' s c o m p l e t e a d j u v a n t by r e p e a t e d e x t r u s i o n t h r o u g h a s y r i n g e e q u i p p e d w i t h a 21G-1 n e e d l e . S u b s e q u e n t i n j e c t i o n s were o f 7.5% g e l b a n d s f r o m 40 t o 50 grams o f f l i e s h o m o g e n i z e d i n s u f f i c i e n t 0.9% N a C l t o g i v e a f i r m homogenate a f t e r r e p e a t e d e x t r u s i o n t h r o u g h a s y r i n g e . The r a b b i t s were b l e d f r o m t h e m a r g i n a l e a r v e i n and 30 t o 40 m i s o f b l o o d were c o l l e c t e d a t 10 t o 14 day i n t e r v a l s . The b l o o d was a l l o w e d t o c l o t f o r 2 h o u r s a t room t e m p e r a t u r e t h e n t h e c l o t a l l o w e d t o c o n t r a c t a t 4° o v e r n i g h t . The c l o t was removed by c e n t r i f u g a t i o n at. 2000 xg f o r 20 m i n u t e s . I m m u n o g l o b u l i n was p r e p a r e d f r o m t h e serum by two s u c c e s s i v e p r e c i p i t a t i o n s a t 35% s a t u r a t i o n w i t h ( N H 4 ) 2 S 0 4 i n 0.1 M T r i s - H C l pH 8.0 a t 4 ° . A f t e r c e n t r i f u g i n g a t 8000 xg f o r 10 m i n u t e s t h e p r e c i p i t a t e was d i s s o l v e d i n a volume o f 0.1 M T r i s - H C l pH 8.0 e q u a l t o t h e s t a r t i n g volume o f serum, t h e n d i a l y s e d a g a i n s t 0.01 M T r i s - H C l pH 8.0 and s t o r e d f r o z e n a t - 8 0 ° . F o r c o u p l i n g t o S e p h a r o s e , i m m u n o g l o b u l i n was f u r t h e r p u r i f i e d by a m o d i f i c a t i o n o f t h e p r o c e d u r e o f F a h e y •dt al. (68) . I m m u n o g l o b u l i n p r e p a r e d as above was d i a l y s e d a g a i n s t 0.01 M s o d i u m p h o s p h a t e pH 7.8 and a p p l i e d t o a c olumn o f D E A E - C e l l u l o s e (2.5 cm x 10 cm) e q u i l i b r a t e d w i t h t h e same b u f f e r . The c o l u m n was washed w i t h 2 t o 3 c o l u m n v o l u m e s o f 0.01M s o d i u m p h o s p h a t e pH 7.8. The f l o w t h r o u g h m a t e r i a l was p o o l e d and p r e c i p i t a t e d a t 40% s a t u r a t i o n w i t h ( N H 4 ) 2 SC» 4, r e d i s s o l v e d i n a volume o f 0.01M s o d i u m p h o s p h a t e pH 7.8 e q u a l t o t h e s t a r t i n g volume o f a n t i - s e r u m , and d i a l y s e d a g a i n s t t h e same b u f f e r . E . I m m u n o d i f f u s i o n O u c h t e r l o n y d o u b l e d i f f u s i o n t e s t s were p e r f o r m e d as d e s c r i b e d b y C h o v n i c k at al. ( 3 ) . D i f f u s i o n p l a t e s were 2.5 cm x 2.5 cm g l a s s s l i d e s c o a t e d w i t h a 2 mm l a y e r o f 1% a g a r o s e i n 0.02M T r i s - H C l pH 8.0. W e l l s w i t h a c a p a c i t y o f 40 A{1 were c u t i n t h e a g a r o s e w i t h a t e m p l a t e and a n t i g e n and a n t i s e r u m p l a c e d i n t h e w e l l s i n a s i n g l e a p p l i c a t i o n . The s l i d e s were i n c u b a t e d a t 5° f o r t h r e e d a y s o r a t room t e m p e r a t u r e f o r 2 d a y s . The s l i d e s were d e v e l o p e d by i m m e r s i n g i n . t h e XDH s t a i n s o l u t i o n d e s c r i b e d p r e v i o u s l y . A f t e r s t a i n i n g f o r XDH a c t i v i t y t h e s l i d e s were soaked o v e r -n i g h t i n w a t e r , t h e n d r i e d . The d r i e d g e l s were s t a i n e d f o r p r o t e i n w i t h 0.25% C o o m a s s i e B l u e i n m e t h a n o l : a c e t i c a c i d : w a t e r (5:1:5) f o r 30 m i n u t e s t h e n d e s t a i n e d . F. I m m u n o a f f i n i t y C h r o m a t o g r a p h y 1. C o u p l i n g t o S e p h a r o s e CL-4B A n t i b o d i e s f r o m 10 ml o f a n t i s e r u m c o n t a i n i n g 3.8 mg p r o t e i n / m l p u r i f i e d by D E A E - c e l l u l o s e c h r o m a t o g r a p h y as d e s c r i b e d above were c o v a l e n t l y l i n k e d t o 25 m l o f S e p h a r o s e CL-4B t h a t h a d b e e n a c t i v a t e d w i t h CNBr e x a c t l y as d e s c r i b e d b y M a r c h zt at, (69) . A f t e r c o u p l i n g , t h e g e l was f i l t e r e d on a c o a r s e , s i n t e r e d - g l a s s f u n n e l , t h e n r e -s u s p e n d e d i n 1M g l y c i n e i n 0.2M s o d i u m b i c a r b o n a t e b u f f e r pH 9.5 and s h a k e n f o r 5 h o u r s t o b l o c k u n r e a c t e d c o u p l i n g g r o u p s . The a n t i b o d y - S e p h a r o s e was t h e n washed s e q u e n t i a l l y w i t h 200 ml 0.1M s o d i u m b i c a r b o n a t e pH 9.5, 400 ml 0.1M s o d i u m a c e t a t e pH 4.0, and 200 ml 0.1M s o d i u m b i c a r b o n a t e pH 9.5, a l l b u f f e r s b e i n g 0.5M N a C l . The g e l was t h e n washed w i t h 400 m i s B u f f e r P and s t o r e d a t 4° as a s u s p e n s i o n i n B u f f e r P. 18 . 2. Immunoadsorption Chromatography For the p u r i f i c a t i o n of XDH, a column of 7ml of antibody-Sepharose was layered on top of a column of Sephadex G-25 medium, (1.4 cm x 4.5 cm antibody-Sepharose and 1.4 cm x 48 cm Sephadex G-25), and washed thoroughly with Buffer B. The XDH preparations were pumped onto the column at a flow rate of 32 ml/hour and the column washed with 50 ml 1M NaCl i n Buffer B and .120 ml 10% gl y c e r o l i n Buffer B. XDH a c t i v i t y was eluted from the column with 2.4M NH4SCN i n 0.01M Tris-HCl pH 8.0, 4mM^-mercaptoethanol and 10% g l y c e r o l . The column was then immediately flushed with Buffer P. 3. E f f e c t of NH^ SCN concentration on XDH a c t i v i t y To determine the eff e c t s of NH^ SCN concentration on XDH a c t i v i t y , A.t/+'*XDH was incubated i n the presence of varied concentrations of NH^ SCN at 4°. Stock solutions of twice the desired concentration of NH^ SCN were made up i n 0.1M Tris-HCl pH 8.0, 1.3 mM ^-mercaptoethanol. Equal + 11 volumes of NH4SCN solution and fiy extract (post DEAE-c e l l u l o s e fraction) were mixed and kept on i c e . At desired i n t e r v a l s , 50 j^l aliquots were taken and the remaining XDH enzyme a c t i v i t y determined using the NAD+ assay as des-cribed for column f r a c t i o n s . G. Synthesis of 9-(p-aminoethoxyphenyl) guanine 1. Synthesis of p-aminophenyl acetamidoethyl ether A mixture of 24.6 grams (0.125 mole) of sodium p - n i t r o p h e n o x i d e and 94 grams (0.5 mole) o f 1 , 2 - d i b r o m o e t h a n e i n 100 m l w a t e r was r e f l u x e d w i t h s t i r r i n g f o r 48 h o u r s . The c o o l e d m i x t u r e was e x t r a c t e d w i t h 100 ml C HC1 3 and t h e e x t r a c t was washed t w i c e w i t h 100 ml and a t h i r d t i m e w i t h 50 ml o f 5% NaOH. The C H C 1 3 e x t r a c t was d r i e d o v e r MgS0 4, t h e n t h e CHClg was removed i n a r o t a r y e v a p o r a t o r . The h o t r e s i d u a l o i l was d i s s o l v e d i n 25 m l o f e t h a n o l , t h e n c h i l l e d and t h e c r u d e p r o d u c t c o l l e c t e d by f i l t r a t i o n . The c r u d e p r o d u c t o was d i s s o l v e d i n 25 ml e t h a n o l , t r e a t e d w i t h N o r i t A a t 55, , and f i l t e r e d h o t t o remove t h e N o r i t A. The m a t e r i a l was r e c r y s t a l l i z e d two t i m e s f r o m e t h a n o l ( I , F i g u r e 1 ) . Y i e l d was 11 grams, m.p. 6 3 - 6 4 ° . A m i x t u r e o f 11 grams o f t h e 2 - b r o m o e t h y l - p -n i t r o p h e n y l e t h e r and 7 grams o f h e x a m e t h y l e n e - t e t r a m i n e i n 100 ml C HC1 3 was r e f l u x e d w i t h s t i r r i n g f o r 100 h o u r s , and t h e p r o d u c t s e p a r a t e d o u t . The p r o d u c t was c o l l e c t e d by f i l t r a t i o n , washed w i t h C HC1 3 and d i s s o l v e d i n 16 ml w a t e r , 80 m l e t h a n o l and 20 m l 1 2 N HC1, t h e n l e t s t a n d f o r 72 h o u r s . The p r e c i p i t a t e d NH 4C1 was removed by f i l t r a t i o n and t h e s o l v e n t removed :^yv v a c u o . The r e s i d u e was d i s s o l v e d i n 100 ml C H C1 3 and 16.2 grams o f t r i e t h y l a m i n e was added and t h e s o l u t i o n h e a t e d t o 5 0 ° . Then 8.2 grams o f a c e t i c a n h y d r i d e was added and t h e c o o l e d s o l u t i o n was washed t h r e e t i m e s w i t h 100 ml o f w a t e r and d r i e d o v e r MgSO^. The C HC1 3 was removed i n v a c u o a n d t h e r e s i d u e d i s s o l v e d i n b e n z e n e , t r e a t e d w i t h N o r i t A, f i l t e r e d a n d c o n c e n t r a t e d i n v a c u o . R e c r y s t a l l i z a t i o n f r o m b e n z e n e y i e l d e d 2.6 grams, mp. 1 1 8 - 1 1 9 ° ( I I I , F i g u r e 1 ) . F i g u r e 1. O u t l i n e o f t h e s y n t h e s i s o f 9-(p a m i n o e t h o x y p h e n y l ) g u a n i n e f r o m s o d i u m p -n i t r o p h e n o x i d e and 2 - a m i n o - 4 - c h l o r o - 5 - p h e n y l a z o - 6 - p y r i m i d i n o l . N O , N O , N O , O'Ntf BrCH 2 CH 2 Br H NH-. (f\\ ^ C H , C H 2 O H / H C I l/Y I 0 C H 4 C H 2 B r H*/Pt 0 II NO, ca 0 C H 2 C H 2 N H C C H 3 m 0 II 0 C H 2 C H 2 N H C C H 2 J J J O C H 2 C H 2 N H 2 H ( C H 3 C H 2 ) 3 N ( C H 3 C 0 ) 2 £ > O H N ^ w N = N C 6 H 5 f , v HN 2 O H N ^ N - - N C 6 H ^ ¥1 H C O O H Zn 0 II N - ^ Y N H C H ~2K IX - N H N H N ^ N ^ N 6N H C I H ^ N ^ ^ N / " N 2EE O C H 2 C H 2 N H 2 R H C O N H , H C O O H F i g . 1 The a c e t a m i d o e t h y l - p - n i t r o p h e n y l e t h e r was t h e n d i s s o l v e d i n 100 ml a b s o l u t e e t h a n o l and s t i r r e d o v e r PtO a t 2.5 p s i g p r e s s u r e u n t i l r e d u c t i o n was c o m p l e t e . The p l a t i n u m was removed by f i l t r a t i o n and t h e e t h a n o l e v a p o r -a t e d In vacuo. R e c r y s t a l l i z a t i o n f r o m t o l u e n e y i e l d e d 1.9 grams p - a m i n o p h e n y l a c e t a m i d o e t h y l e t h e r (IV, F i g u r e 1 ) . 2. S y n t h e s i s o f 9 - ( p - a m i n o e t h o x y p h e n y l ) g u a n i n e 2 - a m i n o - 4 - c h l o r o - 5 - p h e n y l a z o - 6 - p y r i m i d i n o l T (V, F i g u r e 1) was p r e p a r e d f r o m 2 - a m i n o - 4 - c h l o r o - 6 - p y r i m i d i n o 1 and a n i l i n e as d e s c r i b e d by Boon and L e i g h ( 7 0 ) . A s o l u t i o n o f 1.6 grams p - a m i n o p h e n y l - a c e t a m i d o -e t h y l e t h e r (IV) and 1.97 grams 2 - a m i n o - 4 - c h l o r o - 5 - p h e n y l a z o -6 - p y r i m i d i n o l (V) i n 25 grams a b s o l u t e e t h a n o l was r e f l u x e d w i t h s t i r r i n g f o r 6 h o u r s . The p r e c i p i t a t e d p r o d u c t (VI) was f i l t e r e d and washed w i t h e t h a n o l u n t i l t h e w a s h i n g s were l i g h t y e l l o w . The w e t f i l t e r c a k e was s u s p e n d e d i n 30 m i s 98% f o r m i c a c i d , b o i l e d and Zn d u s t added g r a d u a l l y u n t i l t h e s o l u t i o n b l e a c h e d w h i t e , and was b o i l e d g e n t l y f o r an a d d i t i o n a l 30 m i n u t e s . The c o o l e d s o l u t i o n was f i l t e r e d t o remove Zn a n d Zn s a l t s and t h e e t h a n o l was removed -in v a c u o . F i f t y ml o f i c e w a t e r was added t o t h e t h i n s y r u p , and l e f t t o s t a n d a t 4 ° f o r c o m p l e t e p r e c i p i t a t i o n o f t h e p r o d u c t ( V I I ) . The p r e c i p i t a t e was f i l t e r e d and washed w i t h w a t e r , t h e n s u s p e n d e d i n 50 m l f o r m a m i d e and 4 ml 98% f o r m i c a c i d and r e f l u x e d f o r 6.5 h o u r s . A f t e r r e f l u x i n g , 100 m l o f i c e w a t e r was added and t h e m i x t u r e l e f t t o s t a n d o v e r n i g h t a t 4 ° . The p r e c i p i t a t e was c o l l e c t e d on a f i l t e r , t h e n s u s p e n d e d i n 6N H C l and h e a t e d on a s t e a m b a t h f o r 3 h o u r s . The p r e c i p i t a t e d p r o d u c t was f i l t e r e d , a nd d i s s o l v e d i n 0.1 N H C l , t h e n p r e c i p i t a t e d by n e u t r a l i z a t i o n w i t h 0.1 N NH^OH and c o l l e c t e d by c e n t r i f u g a t i o n a t 12,000 xg f o r 10 m i n u t e s and washed t w i c e w i t h 0.01N NH^OH. The c o l l e c t e d p r e c i p i t a t e was d r i e d by l y o p h i l i z a t i o n f r o m 0.01 N NH^OH. Y i e l d was 0.73 grams o f 9 - ( p - a m i n o e t h o x y p h e n y l ) g u a n i n e ( I X , F i g u r e 1 ) . The l y o p h i l i z e d p r o d u c t r a n as a s i n g l e s p o t on s i l i c a g e l i n 3:5 C H C l 3 : C H 3 O H . 3. P r e p a r a t i o n o f 9 - ( p - a m i n o e t h o x y p h e n y l ) g u a n i n e - S e p h a r o s e 6B  A s l u r r y o f 4 m l p a c k e d volume o f S e p h a r o s e 6B i n 4 m l w a t e r and a n e q u a l volume o f 2M Na 2C02 was s t i r r e d s l o w l y on an i c e b a t h u n t i l c o l d . The r a t e o f s t i r r i n g was i n c r e a s e d and 0.4 grams CNBr d i s s o l v e d i n 0.2 ml a c e t o n i t r i l e was a d d e d a l l a t o n c e . A f t e r s t i r r i n g r a p i d l y f o r 2 m i n u t e s , t h e s l u r r y was f i l t e r e d on a c o a r s e s i n t e r e d g l a s s f u n n e l , and washed w i t h c o l d d i s t i l l e d w a t e r , f o l l o w e d b y c o l d d i m e t h y l - f o r m a m i d e . The g e l was f i l t e r e d t o a m o i s t c a k e , t h e n added t o a s o l u t i o n o f 120 mg o f 9 - ( a m i n o e t h o x y p h e n y l ) -g u a n i n e i n 10 m l d i m e t h y l - f o r m a m i d e and 15 ml d i m e t h y l -s u l f o x i d e , s h a k e n i n a p l a s t i c b o t t l e f o r 24 h o u r s a t 4 ° , t h e n a f u r t h e r 24 h o u r s a t room t e m p e r a t u r e . The g e l was t h e n f i l t e r e d , washed w i t h 500 mis 0.05M s o d i u m p h o s p h a t e pH 4.5 and s t o r e d i n 0.05M s o d i u m p h o s p h a t e pH 4.5 w i t h 0.03% NaN-,. 24. H. P r e p a r a t i o n o f B l u e D e x t r a n - S e p h a r o s e CL-4B . B l u e D e x t r a n - S e p h a r o s e was p r e p a r e d e s s e n t i a l l y as d e s c r i b e d by Thompson . i t al, ( 5 1 ) . A s l u r r y o f 40 m l S e p h a r o s e CL-4B i n 80 m i s 2 M N a 2 C C » 3 was s t i r r e d r a p i d l y on an i c e b a t h , and 4 grams CNBr d i s s o l v e d i n 2 m l a c e t o n i t r i l e was added a l l a t o n c e . A f t e r 2 m i n u t e s t h e g e l was f i l t e r e d i n a c o a r s e g l a s s f i l t e r and washed w i t h 250 m l c o l d 0.1M N a H C 0 3 - N a 2 C 0 3 pH 9.5 t h e n f i l t e r e d t o a m o i s t c a k e . The a c t i v a t e d g e l was i m m e d i a t e l y a d d e d t o a s o l u t i o n o f 1.0 gram B l u e D e x t r a n 2000 d i s s o l v e d i n 40 mis 0.2 N a H C 0 3 - N a 2 C 0 3 pH 9.5 and s h a k e n f o r 24 h o u r s a t 4 ° . The g e l was t h e n washed w i t h 200 m i s 0.1 M N a H C 0 3 - N a 2 C 0 3 pH 9.5, 500 m i s 0.5M N a C l , and 200 mis 0.2 M N a H C 0 3 ~ N a 2 C 0 3 pH 9.5, 100 m l B u f f e r P , t h e n s u s p e n d e d as a 1:1 s l u r r y i n B u f f e r P. 3 U s i n g t h e e x t i n c t i o n c o e f f i c i e n t o f 13.6 x 10 M cm"''" a t 610 nm f o r t h e c h r o m o p h o r e o f B l u e D e x t r a n ( 5 2 ) , t h e c o n c e n t r a t i o n o f t h e d y e i n t h e B l u e D e x t r a n -S p e h a r o s e was d e t e r m i n e d t o be 36 I . P r o t e i n D e t e r m i n a t i o n P r o t e i n was d e t e r m i n e d by t h e method o f Lowry e.t al ( 7 1 ) , u s i n g b o v i n e serum a l b u m i n a s a s t a n d a r d . RESULTS AND DISCUSSION A. I m m u n o a f f i n i t y C h r o m a t o g r a p h y 1. E f f e c t o f NH^SCN- c o n c e n t r a t i o n on enzyme a c t i v i t y C h a o t r o p i c s a l t s , s u c h as KI (1) and NH^SCN (59) h a v e b e e n u s e d t o e l u t e a n t i g e n s f r o m columns o f i m m o b i l i z e d a n t i b o d i e s . However i t has a l s o b e e n shown t h a t s u c h a g e n t s i n a c t i v a t e c h i c k e n l i v e r XDH and m i l k x a n t h i n e o x i d a s e b y c a u s i n g t h e d i s s o c i a t i o n o f the;FAD c o f a c t o r (26,27) and t h a t K I i n a c t i v a t e s Vio&ophlla. XDH (1) p r e s u m a b l y i n t h e same way. The e f f e c t s o f i n c r e a s i n g NH^SCN c o n c e n t r a t i o n on XDH a c t i v i t y i s shown i n F i g u r e 2. The enzyme a c t i v i t y i s n o t g r e a t l y a f f e c t e d a t NH 4SCN c o n c e n t r a t i o n s below 2M, w h i l e i n c r e a s i n g t h e c o n c e n t r a t i o n f r o m 2M t o 3M r e s u l t s i n r a p i d l o s s o f enzyme a c t i v i t y . A n d e r s o n zt at. (59) f o u n d t h a t NH 4SCN was an e f f e c t i v e s a l t f o r t h e d i s s o c i a t i o n o f a n t i b o d y - a n t i g e n c o m p l e x e s i n i m m u n o a f f i n i t y c h r o m a t o g r a p h y . They r e p o r t e d t h a t 3M NH 4SCN c o m p l e t e l y e l u t e d a n t i g e n f r o m an a n t i b o d y c o l u m n , w h i l e 2.5 M NH 4SCN was o n l y s l i g h t l y l e s s e f f e c t i v e . Thus u s i n g a s low a c o n c e n t r a t i o n o f NH 4SCN as p o s s i b l e f o r t h e e l u t i o n o f bound XDH w i l l g r e a t l y i n c r e a s e t h e r e t e n t i o n o f a c t i v i t y , p r o v i d e d t h e e l u t e d enzyme i s i m m e d i a t e l y d e s a l t e d . 2. I m m u n o a f f i n i t y c h r o m a t o g r a p h y C o u p l i n g t h e r a b b i t i m m u n o g l o b u l i n t o S e p h a r o s e CL-4B w i t h CNBr y i e l d e d an e f f e c t i v e s u p p o r t f o r i m m u n o a f f i n i t y F i g u r e 2. E f f e c t o f NH 4SCN c o n c e n t r a t i o n on XDH a c t i v i t y . XDH f r o m n.y+ 7 ? 1409N a f t e r h e a t i n g a t 6 0 ° f o r 5 m i n u t e s was i n c u b a t e d w i t h NH^SCN a t 0 ° . A f t e r 10 m i n u t e s , XDH a c t i v i t y was measured by f o l l o w i n g t h e r e d u c t i o n o f NAD + a t 340 nm as d e s c r i b e d i n Methods. V , r e a c t i o n v e l o c i t y i n o t h e a b s e n c e o f NH 4SCN. V^, r e a c t i o n v e l o c i t y i n t h e p r e s e n c e o f NH.SCN. F i g . 2 c h r o m a t o g r a p h y o f XDH. F o l l o w i n g t h e p r o c e d u r e as d e s c r i b e d i n M e t h o d s , a g e l c o n t a i n i n g 1.2 mg o f p r o t e i n p e r m l o f p a c k e d S e p h a r o s e CL-4B was o b t a i n e d . I n t h e f i r s t a t t e m p t s a t i m m u n o a f f i n i t y c h r o m a t o -g r a p h y o f XDH, 3M KI was u s e d t o e l u t e t h e bound XDH f r o m a s m a l l (1.4 cm x 7 cm) column o f a n t i b o d y - S e p h a r o s e , and t h e e l u t e d XDH t h e n d e s a l t e d on a col u m n o f Sephadex G-25 medium. U s i n g t h i s p r o c e d u r e , t h e r e c o v e r y o f XDH a c t i v i t y was v e r y p o o r as t h e t i m e r e q u i r e d t o e l u t e t h e column and t h e n remove t h e s a l t was t o o l o n g . The need f o r r a p i d d e s a l t i n g was met by l a y e r i n g t h e a n t i b o d y - S e p h a r o s e on t o p o f a column o f Sephadex G-25 medium. The bound XDH was e l u t e d a s a s h a r p peak and was i m m e d i a t e l y d e s a l t e d on t h e Sephadex G-25. The p r o f i l e o f a t y p i c a l p u r i f i c a t i o n i s shown i n F i g u r e 3. U s i n g t h i s c o l u m n g e o m e t r y , r e c o v e r i e s o f 24% and 60% o f t h e a p p l i e d XDH a c t i v i t y were o b t a i n e d w i t h 3M NH 4SCN and 2.4 M NH 4SCN r e s p e c t i v e l y . When e l e c t r o p h o r e s e d on 5% SDS-P0 4 g e l s ( 7 2 ) , XDH p u r i f i e d b y i m m u n o a f f i n i t y c h r o m a t o g r a p h y shows v a r i a b l e amounts o f two h i g h m o l e c u l a r w e i g h t b a n d s . I n t h e g e l s c a n shown i n F i g u r e 4 o n l y one m a j o r b a n d i s p r e s e n t , however o t h e r p r e p a r a t i o n s o f XDH h a v e shown two h i g h m o l e c u l a r w e i g h t p o l y p e p t i d e s . The v a r i a b i l i t y i n t h e amounts o f t h e two p o l y p e p t i d e s s u g g e s t s a v a r i a b l e d e g r e e o f ^ p r o t e o l y t i c d e g r a d a t i o n d u r i n g s t o r a g e and p u r i f i c a t i o n . E v i d e n c e t h a t t h e s m a l l e r component was a p r o d u c t o f p r o t e o l y t i c d e g r a d a -t i o n d u r i n g t h e i n i t i a l s t e p s o f t h e f r a c t i o n a t i o n was + 4 F i g . 3. P u r i f i c a t i o n o f Ky 14Q9H XDH on a n t i b o d y -S e p h a r o s e . A h e a t - t r e a t e d and d i a l y s e d e x t r a c t , + 4 p r e p a r e d a s d e s c r i b e d i n Methods, f r o m 64 g o f fiy X.4Q9W i n a volume o f 48 m i s was a p p l i e d t o a 1.4 x 4.5 cm column o f a n t i b o d y - S e p h a r o s e CL-4B w h i c h had b e e n l a y e r e d on t o p o f a 1.4 x 48 cm column o f Sephadex G-25 medium. The column was t h e n washed s u c c e s s i v e l y w i t h 50 m i s o f 1 M N a C l i n 0.01 M T r i s -H C l pH 8.0, 4 mM y 9 - m e r c a p t o e t h a n o l , and 120 m i s 0.01 M T r i s - H C l pH 8.0, 4 mM y # - m e r c a p t o e t h a n o l , 10% g l y c e r o l , t h e n e l u t e d w i t h 40 m i s o f 2.4 M NH 4SCN i n 0.1 M T r i s - H C l pH 8.0, 4 mM y S - m e r c a p t b e t h a n o l , 10% g l y c e r o l . XDH a c t i v i t y was a s s a y e d b y m e a s u r i n g t h e r e d u c t i o n o f NAD + a t 340 nm as d e s c r i b e d i n M e t h o d s . F i g . 3 F i g u r e 4. S c a n o f a 5% SDS-PO^ p o l y a c r y l a m i d e +4 g e l o f >ty JL409H, XDH a f t e r p u r i f i c a t i o n on a n t i -b o d y - S e p h a r o s e CL-4B. 0.2 25 + 0.200 0.175 0.1130 + O i n 10 0.12! O.iOOf 0.075 + 0.050 0.025 DISTANCE OF MIGRATION, cm. OJ F i g . 4 p r e s e n t e d by Edwards zt at,(73). P u r i f i e d r a t l i v e r XDH s i m i l a r i l y shows a p o l y -p e p t i d e o f 130,000 m o l e c u l a r w e i g h t a f t e r t r e a t m e n t w i t h t r y p s i n (16, 2 2 ) . The " c l i p p e d " XDH r e t a i n e d i t s a n t i g e n i c s i t e s as i t was p r e c i p i t a t e d b y a n t i b o d i e s t o i n t a c t r a t l i v e r XDH. The b e h a v i o r o f t h e VJi®&oph<lZa. a n d r a t l i v e r enzymes s u g g e s t s t h a t t h e y a r e m u l t i d o m a i n p r o t e i n s . B. I m m u n o d i f f u s i o n I n F i g u r e 5 a d i f f u s i o n p l a t e i s shown where + 4 fiy t.409H XDH p u r i f i e d b y iiimuno^fSiriibfey c h r o m a t o g r a p h y + 4 ( w e l l s 1 and 4) , a c r u d e h e a t - t r e a t e d ty X.4Q9W e x t r a c t ( w e l l s 3 and 5) and homogenate s u p e r n a t a n t s f r o m two x - r a y S 41 i n d u c e d - t y - n u l l o m u t a n t s ity ( w e l l 2) and ny ( w e l l 6) were d i f f u s e d a g a i n s t 1:5 d i l u t e d a n t i s e r u m . The p u r i f i e d XDH g a v e o n l y one p r e c i p i t i n linejWhich c o n t a i n e d XDH + 4 a c t i v i t y . H e a t t r e a t e d e x t r a c t o f fiy Z4 09H gave two p r e c i p i t i n l i n e s , t h e o u t e r p r e c i p i t i n l i n e c o n t a i n e d XDH a c t i v i t y and was i d e n t i c a l t o t h e X D H - a c t i v e p r e c i p i t i n l i n e o f t h e p u r i f i e d XDH. The s e c o n d i n n e r p r e c i p i t i n - l i n e d i d n o t c o n t a i n XDH a c t i v i t y , y e t showed a r e a c t i o n o f p a r t i a l i d e n t i t y w i t h t h e a n t i g e n o f t h e p u r i f i e d XDH. S i m i l a r r e s u l t s were o b t a i n e d w i t h e x t r a c t s o f H.y+^ 1 4 Q 9 U . The two f i y - m x l l o m u t a n t shown h e r e g a v e one p r e c i p i t i n l i n e , w h i c h d i d n o t c o n t a i n XDH a c t i v i t y y e t showed i d e n t i t y w i t h t h e n o n - s t a i n i n g p r e c i p i t i n l i n e o f F i g u r e 5. I m m u n o d i f f u s i o n o f Vh.obophi.La. e x t r a c t s + 4 i n 1% a g a r o s e . 1 & 4, t y JL409W e x t r a c t p u r i -4 7 f i e d by i m m u n o a d s o r p t i o n c h r o m a t o g r a p h y . 2, Ay + 4 . homogenate s u p e r n a t a n t . 3 & 5, fiy 4.409H a f t e r o & h e a t i n g a t 60 f o r 5 m i n . 6, fiy homogenate s u p e r -n a t a n t . The p l a t e was s t a i n e d s u c c e s s i v e l y f o r XDH a c t i v i t y and f o r p r o t e i n . The p r e c i p i t i n l i n e s i n d i c a t e d by t h e a r r o w d i d n o t s t a i n f o r XDH a c t i v i t y . F i g . 5 36. + 4 the crude Ky 4.409H extract and p a r t i a l i d e n t i t y with the p u r i f i e d XDH. Similar r e s u l t s were obtained with A.y-nullo mutants 1, 5, 26, 201, 203, 204 and 502. A reaction of p a r t i a l i d e n t i t y indicates that one antigen possesses some antigenic s i t e s of the second antigen while the second antigen possesses a l l of the a n t i -genic determinants of the f i r s t antigen. Reactions of p a r t i a l i d e n t i t y were obtained by Reed ztalXIA)when two fragments of bovine serum albumin produced by limited p r o t e o l y s i s , and i n t a c t bovine serum albumin were di f f u s e d against anti-BSA-antiserum. They concluded that bovine serum albumin was a multi-domain protein. The immunodiffusion r e s u l t s observed here also suggest that XDH i s subject to some limited proteolysis during storage and p u r i f i c a t i o n . The antigen that does not stain for XDH a c t i v i t y seems to be l o s t on immunoaffinity chromatography and may represent degraded XDH. The reaction of p a r t i a l i d e n t i t y between the non-active antigen i n the /tt/-nullo extracts and p u r i f i e d , active XDH indicates that the A.t/-nullo strains tested contain cross-reacting material to XDH. This could be XDH monomers or inactive dimer. Proof of the antigen being cross-reacting material to XDH, that i s ny gene product, would require testing a known CRM s t r a i n such as a homozygous Ky deletion. Such a s t r a i n was not available for tes t i n g . C. Kinetics 1. Determination of Kinetic Constants The comparison of the k i n e t i c constants of the XDH produced by *y+4l409H and Hy*^'1409H i s of i n t e r e s t as i t has been suggested that the 4.409 s i t e i s involved i n the regulation of the iy-locus, rather than a s t r u c t u r a l a l t e r -ation of the Jiy gene product. I t would have been preferable +4 . +4 + 11 ' to compare XDH from ny and ny 1409H or from iy 1409H and n.y+^ 1409H, however the two stocks hy+*i409H and f i y + ^ 1409H were not available. The two i s o a l l e l e s used, + 4 +11 fiy and n.y are e l e c t r o p h o r e t i c a l l y i d e n t i c a l , and have the same thermolability at 60° as shown by Chovnick nt al. (3) . + 4 +11 For k i n e t i c analysis, XDH from h.y and ny was p u r i f i e d by immunoaffinity chromatography and the enzyme a c t i v i t y assayed as described i n Methods. When the data were plotted on Lineweaver-Burke +4 • +11 double r e c i p r o c a l p l o t s , both Ky 4.409H and h.y JL409U gave plots of p a r a l l e l l i n e s as shown i n Figures 6A, C, E, G. As described i n Segel (75) such double r e c i p r o c a l plots are obtained when an enzyme has a ping-pong b i - b i reaction mechanism, where a product i s released between the addition of two substrates. A s i m p l i f i e d reaction scheme written i n the notation of Cleland (76) for the reaction of XDH with xanthine and NAD+ i s shown i n Figure 7. In the absence of product, the v e l o c i t y equation for a Ping Pong B i B i reaction mechanism i s given by F i g u r e 6. D o u b l e r e c i p r o c a l p l o t s 1/ +4 v e r s u s ' [ s u b s t r a t e ] f o r XDH f r o m Ky 1409H and fiy+^ 1409U p u r i f i e d b y immuno-+ 4 a d s o r p t i o n c h r o m a t o g r a p h y . A-D, n.y <L40 9H. A, v e r s u s [ x a n t h i n e ] , NAD + c o n -c e n t r a t i o n s were 90 >c<M, 60 MM, 45,6(M, 18 x(M and l l . S ^ M . B, r e p l o t o f a p p a r e n t ^ V m a x f r o m A v e r s u s ^ [NAD +] . C, " ^ v v e r s u s [NAD +] , x a n t h i n e c o n c e n t r a t i o n s were 91>(M, 45.5x(M, 27.3x(M and 18.2>i<M. D, r e p l o t o f a p p a r e n t "^Vmax f r o m C v e r s u s 1/ [ x a n t h i n e ] . E-H fty+U1409H. E, ^ v v e r s u s " ^ [ x a n t h i n e ] , NAD + c o n c e n t r a t i o n s were as i n A. F, r e p l o t o f a p p a r e n t "^V max f r o m E v e r s u s 1 / / [ N A D + ] , G, " ^ v v e r s u s ^^[NAD +] , x a n t h i n e c o n c e n t r a t i o n s w ere as i n C. H. r e p l o t o f a p p a r e n t max f r o m G v e r s u s [ x a n t h i n e ] . 39. 3.0 5.0 y/[NAD*] , M " ' x 1 0 " 4 10 2.0 3.0 4.0 5.0 1 /[XANTHINE] , M - 1 x 1 0 - 4 0.2 0.4 0.6 0.8 ^/[XANTHINE] , M " ' x 1 0 " 4 2.0 6.0 10.0 14.0 V[NAD*], M - ' x 1 0 " 4 2.0 6.0 10.0 14.0 1 / [ N / » D + ] , M'1 x 1 0 " 4 2.0 4.0 6.0 8.0 ^/[XANTHINE] , M - ' x I O " 4 F i g . 6 F i g u r e 7. P i n g - p o n g r e a c t i o n s e q u e n c e f o r XDH. XANTHINE URIC ACID NAD NADH XDH°X XDH°- X ^  XDH r6-dUA X D H f e d X D H N A D —XDH°- NADH. XDH o x F i g . 7 42. E q u a t i o n 1, where A and B a r e t h e two s u b s t r a t e s . v = [A] [B] E q u a t i o n 1 Vmax K m B I A K + ^ A t B ] + [ A ] [ B ] " I n r e c i p r o c a l f o r m , s h o w i n g e a c h s u b s t r a t e as t h e v a r i e d s u b -s t r a t e , t h e v e l o c i t y e q u a t i o n becomes: 1 Km, 1 v A ... + (1 + Knig ) E q u a t i o n 2 V x W Vmax ( : [BL ) (A i s v a r i e d ) h~ ~ ^ 1 + _ 1 _ (1 + Km A ) E q u a t i o n 3 TAT V Vmax TBT Vmax ( -rrr ) (B i s v a r i e d ) The s l o p e o f t h e p l o t 1 / / v v s 1 /'[A] a t f i x e d B i s i n d e p e n d e n t o f B , w h i l e t h e " ' "A f-axis i n t e r c e p t i s an a p p a r e n t ^ ^ v m a x w h i c h i s d e p e n d e n t on t h e v a l u e s o f Knig and [B] as shown b y E q u a t i o n 4 _1 = __1 + m^B_ 1 E q u a t i o n 4 Vmax Vmax V~ IBJ app max The v a l u e o f Km^ c a n t h e r e f o r e be d e t e r m i n e d by r e p l o t t i n g t h e " ^ v - a x i s i n t e r c e p t s f r o m a ^ v v s . ^ [ A ] p l o t a g a i n s t V . Km A c a n be e v a l u a t e d i n a s i m i l a r manner. The p r i m a r y p l o t s o f 1 / / v v s . ''"/[xanthine] and r e p l o t s o f 1 / /Vmax v s . 1 / ' [ N A D t ] f o r fiy+4l409H and Ky*^i4 09H app J a r e shown i n F i g u r e 6A, B and 6E, F . The p r i m a r y p l o t s o f ^Ar v s . ^ t N A D ] and r e p l o t s o f ^Vmax v s . ^ [ x a n t h i n e ] f o r app + 4 + 11 h.y 1409H and fiy 14Q9H a r e shown i n F i g u r e 6 C , D and 6G,H. The d a t a w i t h h y p o x a n t h i n e a s s u b s t r a t e a r e n o t shown. The v a l u e s f o r K m ( x a n t h i n e ) , K m ( h y p o x a n t h i n e ) , and K m ( N A D + ) d e t e r m i n e d i n t h i s way a r e g i v e n i n T a b l e 1. TABLE 1 K v a l u e s f o r XDH m f r o m iy+4i409Haid h.y+U14Q9U w i t h x a n t h i n e , h y p o x a n t h i n e and NAD + S u b s t r a t e K , m o l e s / 1 m Ky+414Q9H Ky+U1409H x a n t h i n e 2.1 x 1 0 " 5 2.4 x 1 0 ~ 5 h y p o x a n t h i n e 1.7 x 1 0 ~ 5 1.6 x I O - 5 NAD* 2.6 x 1 0 ~ 5 4.0 x 1 0 ~ 5 + 4 + 11 The two i s o a l l e l e s , ny 1409H and ny 4.4 09N h a v e t h e same K m v a l u e s f o r s u b s t r a t e s , c o n s e q u e n t l y t h e i n c r e a s e d XDH enzyme + 4 a c t i v i t y i n n.y A,409H f l i e s i s n o t due t o a d i f f e r e n c e i n a f f i n i t y f o r s u b s t r a t e . P r e v i o u s l y Y e n and G l a s s m a n (31) examined t h e k i n e t i c b e h a v i o u r o f XDH f r o m f o u r e l e c t r o p h o r e t i c v a r i a n t s o f t h e <*t/-locus. They r e p o r t e d K m v a l u e s f o r x a n t h i n e r a n g i n g f r o m 1.8 x 10 5M t o 2.5 x 10 5M and K m v a l u e s f o r NAD + r a n g i n g f r o m 2.5 x 10~ 5M t o 4.0 x 10~ 5M. P i n g Pong r e a c t i o n mechanisms have a l s o b e e n r e p o r t e d f o r t u r k e y (18) and c h i c k e n (17) x a n t h i n e d e h y d r o -g e n a s e s , as w e l l as b o v i n e x a n t h i n e o x i d a s e ( 1 9 ) . 44. 2. I n h i b i t i o n by 9 - ( p - a m i n o e t h o x y p h e n y l ) g u a n i n e The work o f B a k e r and c o - w o r k e r s l e d t o t h e s y n t h e s i s o f s e v e r a l d e r i v a t i v e s o f 9 - p h e n y l g u a n i n e w h i c h were i n h i b i t o r s o f b o v i n e m i l k x a n t h i n e o x i d a s e (33-36, 7 7 ) . One compound 9 - ( p - a m i n o e t h o x y p h e n y l ) g u a n i n e (compound IX, F i g u r e 1) was r e p o r t e d t o be a n e f f e c t i v e l i g a n d f o r t h e a f f i n i t y c h r o m a t o g r a p h y o f b o v i n e m i l k x a n t h i n e o x i d a s e and r a t l i v e r g u a n i n e d e a m i n a s e and x a n t h i n e d e h y d r o g e n a s e ( 3 5 ) . 9 - ( p - a m i n o e t h o x y p h e n y l ) g u a n i n e was s y n t h e s i z e d b y t h e methods o f B a k e r and Wood (36) as d e s c r i b e d i n M e t h o d s . C h r o m a t o g r a p h y on s i l i c a g e l i n 3:5 CHC1 3:CH 30H showed a s i n g l e s p o t . + 4 The i n h i b i t i o n o f fiy 4.40 9H XDH was d e t e r m i n e d as d e s c r i b e d i n M e t h o d s . As shown i n F i g u r e 8, p l o t t i n g " ^ v v s . ^ [ h y p o x a n t h i n e ] a t d i f f e r e n t , f i x e d c o n c e n t r a t i o n s o f t h e i n h i b i t o r y i e l d s a s e r i e s o f l i n e s t h a t i n t e r s e c t t o t h e l e f t o f t h e ^ v - a x i s a n d above t h e [ h y p o x a n t h i n e ] - a x i s . As d i s c u s s e d by S e g e l ( 7 5 ) , an i n h i b i t o r o f a m u l t i - s u b s t r a t e enzyme a f f e c t s t h e "Vv-axis i n t e r c e p t i f i t and t h e v a r i e d s u b s t r a t e combine w i t h d i f f e r e n t f o r m s o f t h e enzyme, and s a t u r a t i o n w i t h t h e v a r i e d s u b s t r a t e c a n n o t o v e r -come t h e i n h i b i t i o n . S i m i l a r i l y , an i n h i b i t o r a f f e c t s t h e s l o p e o f a d o u b l e r e c i p r o c a l p l o t i f i t and t h e v a r i e d s u b -s t r a t e combine r e v e r s i b l y w i t h t h e same enzyme f o r m o r w i t h d i f f e r e n t f o r m s t h a t a r e s e p a r a t e d i n t h e r e a c t i o n s e q u e n c e by f r e e l y r e v e r s i b l e s t e p s . Thus t h e i n h i b i t i o n o f XDH by F i g u r e 8. I n h i b i t i o n o f XDH b y 9 - ( p - a m i n o e t h o x y -p h e n y l ) g u a n i n e . D o u b l e r e c i p r o c a l p l o t o f v e r s u s " ^ [ h y p o x a n t h i n e ] i n t h e p r e s e n c e o f 9 - ( p -+ 4 a m m o e t h o x y p h e n y l ) g u a n i n e f o r XDH f r o m fiy Z409H p u r i f i e d by i m m u n o a d s o r p t i o n c h r o m a t o g r a p h y . NAD c o n c e n t r a t i o n was 2 mM, 9 - ( p - a m i n o e t h o x y p h e n y l ) g u a n i n e c o n c e n t r a t i o n s w e r e O^M, 5.7^M, 7.1x<.M, 8.5x(M, 1 0 . 6 ^ M and 14.2x<M. 9 - ( p - a m i n o e t h o x y p h e n y l ) - g u a n i n e c a n be a c c o u n t e d f o r b y a r e a c t i o n scheme as shown i n F i g u r e 9, t h e v e l o c i t y e q u a t i o n s f o r w h i c h a r e shown i n E q u a t i o n s 5 and 6. _!L_ ' = t H x l E q u a t i o n 5 Vmax 1 = 1 v V_ max K < i + J£!> + Km(HX) ( 1 + K ± 1 ) 1 + K m ( N A D ) [ 1 + 1 + Km(NAD) (1 + TNADT V * i 2 [NAD] Km(Hx) j f l + [ I ] ) 1 V [ K. , j [Hx] max v i l .' [HX] E q u a t i o n 6 The v a l u e o f c a n be r e a d i l y e v a l u a t e d by r e -p l o t t i n g t h e s l o p e o f t h e l i n e s f r o m t h e ^ v v s . ^ [ h y p o x a n t h i n e ] p l o t a g a i n s t [I] as shown i n F i g u r e 10. The [ I ] - a x i s i n t e r c e p t — 6 i s e q u a l t o - K J L I / t h e r e f o r e K i ^ i s e q u a l t o 4.55 x 10 M. The v a l u e f o r K^ 2 c a n be d e t e r m i n e d by s u b s t i t u t i o n o f known v a l u e s f o r V m a x / K m ( H x ) , K m ( N A D ) , [NAD] and t h e v a l u e o f i n t o t h e terms where K^ 2 a p p e a r s . I n t h e r e p l o t o f a x i s i n t e r c e p t f r o m F i g u r e 8 a g a i n s t [I] i n F i g u r e 11, t h e [I] a x i s i n t e r c e p t i s g i v e n by -K. 9 ( 1 + y ^ 0 ^ — ) and t h e s l o p e by " m(NAD) K m ( N A D ) ^ V m a x [ N A D ] K ^ 2 . I n F i g u r e 8 t h e 1 / [ h y p o x a n t h i n e ] c o o r d i n a t e o f t h e i n t e r s e c t i o n p o i n t o f t h e l i n e s i s g i v e n by ^ ( N A D ) ^ Km(Hx) K i 2 ^ N A D ' ' * S u b s t i t u t i n g i n t o t h e s e terms g i v e s v a l u e s f o r K i 2 o f 5.39 x 10~ 7M, 5.77 x 10~ 7M and 5.97 x 10~ 7M, t o g i v e -7 a mean v a l u e f o r K., o f 5.7 x 10 M. i 2 As K^^ and K^ 2 a r e t h e d i s s o c i a t i o n c o n s t a n t s f o r t h e XDH 0 X-AEPG and X D H r e d - A E P G com p l e x e s r e s p e c t i v e l y , t h e i n h i b i t o r i s more t i g h t l y bound t o t h e p a r t i a l l y r e d u c e d XDH t h a n t o o x i d i z e d XDH. A s i m i l a r s i t u a t i o n i s known i n t h e i n h i b i t i o n o f x a n t h i n e o x i d a s e b y a l l o p u r i n o l , a p y r a z o l o -F i g u r e 9. P r o p o s e d i n h i b i t i o n o f XDH by 9 - ( p - a m i n o e t h o x y p h e n y l ) g u a n i n e . I , 9 - ( p -a m i n o e t h o x y p h e n y l ) g u a n i n e . K - j ^ ' d i s s o c i -ox a t i o n c o n s t a n t f o r t h e XDH - I c o m p l e x . K d i s s o c i a t i o n c o n s t a n t f o r t h e XDH - I complex. HYPOXANTHINE XANTHINE NAO NADH XDH ox Ki. X D H ^ H X ^ X D H ^ X X D H red f « d ox XDH - NAD^XDH - NADH XDH ox XDH - I r e d XDH - I F i g . 9 F i g u r e 10. R e p l o t o f t h e s l o p e 1/ f r o m a p l o t o f 1 / / v v e r s u s ^ [ h y p o x a n t h i n e ] ( F i g u r e 8) v e r s u s c o n c e n t r a t i o n o f 9 - ( p - a m i n o e t h o x y p h e n y l ) g u a n i n e . 51 F i g u r e 11. R e p l o t o f t h e a p p a r e n t v m a x f r o m p l o t o f v e r s u s ^ [ h y p o x a n t h i n e ] ( F i g u r e 8) v e r s u s c o n c e n t r a t i o n o f 9 - ( p - a m i n o e t h o x y p h e n y l ) g u a n i n e . es [3,4-d] p y r i m i d i n e ( 3 8 , 3 9 ) . A l l o p u r i n o l b i n d s t o o x i d i z e d -7 x a n t h i n e o x i d a s e w i t h a d i s s o c i a t i o n c o n s t a n t o f 7 x 10 M (78) and u n d e r g o e s a s l o w e n z y m a t i c h y d r o x y l a t i o n i n p o s i t i o n 6. The p r o d u c t , a l l o - x a n t h i n e , c omplexes v e r y t i g h t l y t o t h e enzyme bound molybdenum i n t h e Mo(IV) s t a t e , w i t h a d i s s o c i a -t i o n c o n s t a n t o f 5.4 x 10 "^M ( 3 9 ) . Whether o r n o t t h e 9 - ( p -a m i n o e t h o x y p h e n y l ) g u a n i n e i s h y d r o x y l a t e d b y XDH, t h e b i n d i n g o f an i n h i b i t o r t o a p a r t i a l l y r e d u c e d enzyme, and t h e r e b y p r e v e n t i n g i t s r e o x i d a t i o n , i s known t o o c c u r i n t h e i n h i b i t i o n o f x a n t h i n e o x i d a s e b y a l l o p u r i n o l . D. A f f i n i t y C h r o m a t o g r a p h y The p u r i f i c a t i o n o f XDH by a f f i n i t y c h r o m a t o -g r a p h y was a t t e m p t e d u s i n g 9 - ( p - a m i n o e t h o x y p h e n y l ) g u a n i n e -S e p h a r o s e 6B, NAD^ .-Agarose and B l u e D e x t r a n - S e p h a r o s e CL-4B. 9 - ( p - a m i n o e t h o x y p h e n y l ) g u a n i n e was c o u p l e d t o C N B r - a c t i v a t e d S e p h a r o s e 6B as d e s c r i b e d . An e x t r a c t o f +1 1 H.y Z40 9H a f t e r D E A E - c e l l u l o s e c h r o m a t o g r a p h y was a p p l i e d t o a 5 cm. column o f t h e A E P G - S e p h a r o s e p a c k e d i n a P a s t e u r p i p e t t e i n 50 mM T r i s - H C l pH 7.4. A t t h e same t i m e , m i l k x a n t h i n e o x i d a s e was a p p l i e d t o a s e c o n d c o l u m n . B o t h columns washed w i t h 50 mM T r i s - H C l , pH 7.4 f o l l o w e d by 2 mM h y p o x a n -t h i n e and t h e r e s p e c t i v e enzyme a c t i v i t i e s f o l l o w e d . No XDH a c t i v i t y was bound t o t h e c o l u m n w h i l e 90% o f t h e a p p l i e d x a n t h i n e o x i d a s e a c t i v i t y was bound and t h e n e l u t e d by t h e h y p o x a n t h i n e wash ( d a t a n o t shown) as was r e p o r t e d b y B a k e r +11 and S e i b e n e i c k (35) . The f a i l u r e o f h.y XDH t o b i n d i s p r o b a b l y due t o a g r e a t e r s u s c e p t i b i l i t y t o s t e r i c h i n d r a n c e 55. p o s e d b y t h e S e p h a r o s e s u p p o r t s i n c e XDH has c o n s i d e r a b l e a f f i n i t y f o r t h e f r e e l i g a n d , as h a s b e e n shown. I m m o b i l i z e d NAD + h a s b e e n r e p o r t e d t o be a u s e -f u l s u p p o r t f o r t h e a f f i n i t y c h r o m a t o g r a p h y o f N A D + - s p e c i f i c + 4 d e h y d r o g e n a s e s ( 5 3 , 5 4 ) . XDH f r o m Jiy was f o u n d t o be r e a d i l y bound t o c o m m e r c i a l N A D + - A g a r o s e , b u t was e l u t e d w i t h low s a l t c o n c e n t r a t i o n and was n o t e l u t e d w i t h 0.2 mM NAD +. The b i n d i n g o f XDH t o NAD*-Agarose was c o n c l u d e d t o be due t o i o n i c i n t e r a c t i o n s r a t h e r t h a n b i o l o g i c a l s p e c i f i -c i t y and was n o t p u r s u e d f u r t h e r . C i b a c r o n b l u e F3GA, t h e chromophore o f B l u e D e x t r a n , has b e e n r e p o r t e d t o be an a n a l o g u e o f NAD"*7 (52) , and enzymes t h a t c o n t a i n t h e d i n u c l e o t i d e f o l d a r e r e a d i l y bound t o B l u e D e x t r a n - S e p h a r o s e (50, 5 1 ) . B l u e D e x t r a n 2000 was c o u p l e d t o C N B r - a c t i v a t e d S e p h a r o s e CL-4B as d e s c r i b e d i n M e t h o d s , and t e s t e d f o r i t s a b i l i t y t o b i n d XDH. I n 10 mM T r i s - H c l pH 7.5 as u s e d by Thompson zt al. ( 5 1 ) , none o f t h e a p p l i e d XDH was bou n d t o a column o f B l u e D e x t r a n - S e p h a r o s e . As t h e c o n d i t i o n s u s e d were s u i t a b l e f o r t h e b i n d i n g o f a w i d e v a r i e t y o f enzymes no f u r t h e r a t t e m p t s were made. CONCLUSION X a n t h i n e d e h y d r o g e n a s e f r o m Vto t,opki.la malano-Qai>t(Lh. i s v e r y s i m i l a r t o x a n t h i n e d e h y d r o g e n a s e s f r o m o t h e r s o u r c e s . Homogeneous XDH has a s u b u n i t m o l e c u l a r w e i g h t . o f 150,000. D u r i n g i n i t i a l s t e p s o f f r a c t i o n a t i o n , p r o t e o l y t i c d e g r a d a t i o n g i v e s a c l i p p e d monomer o f 130,000 m o l e c u l a r w e i g h t . 1. P a r t i a l l y p u r i f i e d XDH was i n a c t i v a t e d by NH^SCN. A t c o n c e n t r a t i o n s b e l o w 2M NH 4SCN t h e r e was l i t t l e e f f e c t on enzyme a c t i v i t y . As t h e c o n c e n t r a t i o n i n c r e a s e d f r o m 2M t o 3M XDH was r a p i d l y i n a c t i v a t e d . 2. I m m u n o a d s o r p t i o n c h r o m a t o g r a p h y y i e l d e d e s s e n t i a l l y p u r e XDH. U s i n g 2.4M NH 4SCN t o d i s s o c i a t e t h e bound enzyme, a 60% r e c o v e r y o f enzyme a c t i v i t y was o b t a i n e d . R e c o v e r y o f a c t i v e enzyme was d e p e n d e n t on r a p i d d e s a l t i n g b y p a s s a g e t h r o u g h S e p h a d e x G-25. 3. Enzyme p u r i f i e d by i m m u n o a d s o r p t i o n showed a p i n g pong r e a c t i o n mechanism where t h e r e i s t h e r e l e a s e o f a p r o d u c t between t h e b i n d i n g o f two s u b s t r a t e s . The XDH + 4 + 11 p r o d u c e d b y two a l l e l e s o f 4,409, fiy 4.409H a n d ny 1409H h a d t h e same K m v a l u e s f o r x a n t h i n e , h y p o x a n t h i n e and NAD. T h e r e i s f u r t h e r e v i d e n c e t h a t t h e i n c r e a s e d XDH a c t i v i t y i n 4-409H f l i e s i s n o t due t o a s t r u c t u r a l a l t e r a t i o n o f t h e XDH m o l e c u l e , b u t r a t h e r r e s u l t s f r o m i n c r e a s e d amounts o f XDH p e r f l y . 4. The compound 9 - ( p - a m i n o e t h o x y p h e n y l ) g u a n i n e was a dead-end i n h i b i t o r o f Vno&ophlla. XDH. U n l i H e b o v i n e x a n t h i n e o x i d a s e , t h e Vtio&ophLla. enzyme d i d n o t b i n d t o 9 - t p - a m i n o e t h o x y -p h e n y l ) g u a n i n e - S e p h a r o s e 6B. 5. VtLO&opklla. XDH d i d n o t b i n d t o B l u e D e x t r a n - S e p h a r o s e n o r t o NAD-Agarose. B i n d i n g t o B l u e D e x t r a n i s common t o p r o t e i n s t h a t p o s s e s s t h e d i n u c l e o t i d e f o l d . I t i s a t t r a c t i v e t o s p e c u l a t e t h a t VKoAophLZdsXDK does n o t c o n t a i n t h i s s t r u c t u r e . 58. REFERENCES 1. A n d r e s , R.Y. (1976) E u r J . B i o c h e m . 62, 591-600. 2. G e l b a r t , W., M c C a r r o n , M., & C h o v n i c k , A. (1976) G e n e t i c s 84. 3. C h o v n i c k , A., G e l b a r t , W., M c C a r r o n , M., Osmond, B., C a n d i d o , E.P.M., & B a i l l i e , D.L. (1976) G e n e t i c s 84, 233-255. 4. C h o v n i c k , A., (1966) P r o c . Roy. S o c . London B164, 198-208. 5. 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