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Studies on the 11[Beta]-hydroxylation of deoxycorticosterone Williamson, Denis George 1968

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STUDIES ON THE 113-HYDROXYLATION OF DEOXYCORTICOSTERONE by DENIS GEORGE WILLIAMSON B.Sc, University of B r i t i s h Columbia, 1963 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of Biochemistry We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF May, BRITISH COLUMBIA 1968 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced deg ree at t he 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 ag ree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r ag r ee t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y pu rposes may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n -t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f Biochemistry  The U n i v e r s i t y o f B r i t i s h Co lumb ia Vancouve r 8, Canada Date M a y 2 7 , 1 9 6 8 i A B S T R A C T S t e r o i d h y d r o x y l a s e s a r e members o f a g r o u p o f enzymes t e r m e d " m i x e d f u n c t i o n o x i d a s e s " . T h e s e enzymes c a t a l y z e t h e i n t r o d u c t i o n o f a n a t o m o f m o l e c u l a r o x y g e n i n t o t h e s u b s t r a t e m o l e c u l e c o n c o m i t a n t w i t h t h e o x i d a t i o n o f NADPH. T h i s t h e s i s d e s c r i b e s s t u d i e s c a r r i e d o u t w i t h a s t e r o i d 1 1 3 - h y d r o x y l a s e p r e p a r e d f r o m a n a c e t o n e p o w d e r o f b o v i n e a d r e n a l m i t o c h o n d r i a . T h e c o n v e r s i o n o f d e o x y c o r t i c o s t e r o n e t o c o r t i c o s t e r o n e h a s b e e n e m p l o y e d t o m e a s u r e t h e 1 1 3 - h y d r o x y l a s e a c t i v i t y . T h e modes o f a c t i o n o f two i n h i b i t o r s o f t h e l l g - h y d r o x y l a t i o n r e a c t i o n , n a m e l y , d i c u m a r o l a n d M e t o p i r o n e h a v e b e e n e x a m i n e d as a means o f o b t a i n i n g i n f o r m a t i o n c o n c e r n i n g t h e m e c h a n i s m o f H B - h y d r o x y l a t i o n . K i n e t i c e x a m i n a t i o n o f d i c u m a r o l i n h i b i t i o n o f 1 1 3 - h y d r o x y -l a t i o n i n d i c a t e d t h a t t h i s compound h a d a t l e a s t two i n h i b i t o r y a c t i o n s . A t c o n c e n t r a t i o n s b e l o w 100 u m o l e s / 1 , d i c u m a r o l was a n o n c o m p e t i t i v e i n h i b i t o r o f 1 1 3 - h y d r o x y l a t i o n . A t d i c u m a r o l c o n c e n t r a t i o n s a b o v e 100 u m o l e s / 1 a s e c o n d i n h i b i t o r y a c t i o n became a p p a r e n t . T h i s s e c o n d i n h i b i t i o n c o u l d be g r e a t l y d i -m i n i s h e d b y i n c r e a s i n g t h e s u b s t r a t e c o n c e n t r a t i o n . K i n e t i c e x a m i n a t i o n o f M e t o p i r o n e i n h i b i t i o n o f 113-h y d r o x y l a t i o n i n d i c a t e d t h a t t h i s compound was a c o m p e t i t i v e i n h i b i t o r o f t h e 1 1 3 - h y d r o x y l a s e r e a c t i o n . I n a d d i t i o n M e t o -p i r o n e h a d a h i g h e r a f f i n i t y f o r t h e 1 1 3 - h y d r o x y l a s e s y t e m t h a n d i d s u b s t r a t e d e o x y c o r t i c o s t e r o n e . T h e K^ f o r M e t o -- 7 p i r o n e was 1.0 x 10 m o l e s / 1 w h i l e t h e K ^ f o r d e o x y c o r t i -— 6 c o s t e r o n e was 5.5 x 10 m o l e s / 1 . C y t o c h r o m e P-450 h a s b e e n shown t o be b o t h t h e o x y g e n -a c t i v a t i n g a n d s u b s t r a t e b i n d i n g c o m p o nent o f s t e r o i d h y d r o x y -l a s e s . The i n t e r a c t i o n s o f s t e r o i d s u b s t r a t e , d i c u m a r o l , a n d M e t o p i r o n e w i t h t h i s h e m o p r o t e i n w e r e t h e r e f o r e e x a m i n e d . The a b i l i t y o f c y t o c h r o m e P-450 t o b i n d c a r b o n m o n o x i d e f o r m i n g a c o m p l e x e x h i b i t i n g a n a b s o r p t i o n maximum a t 450 my was e m p l o y e d t o m e a s u r e t h i s c y t o c h r o m e i n t h e l l g - h y d r o x y l a s e p r e p a r a t i o n . C y t o c h r o m e P-450 p r e s e n t i n t h e m i t o c h o n d r i a l a c e t o n e p owder p r e p a r a t i o n was f o u n d t o be u n s t a b l e , u n d e r g o i n g s p o n -t a n e o u s d e c o m p o s i t i o n a t t e m p e r a t u r e s a b o v e 30° C t o c y t o -c hrome P-420, a h e m o p r o t e i n t h a t d o e s n o t f u n c t i o n i n 116-h y d r o x y l a t i o n . H o w e v e r , t h e r a t e a n d e x t e n t o f c y t o c h r o m e P-4 50 d e c o m p o s i t i o n was d i m i n i s h e d by t h e a d d i t i o n o f s t e r o i d s u b s t r a t e , s u g g e s t i n g t h a t s u b s t r a t e was b i n d i n g t o a n d s t a b i -l i z i n g t h e h e m o p r o t e i n . A s i m i l a r s t a b i l i z a t i o n o f c y t o -chrome P-4 50 was p r o d u c e d u p on a d d i t i o n o f M e t o p i r o n e a n d o f l o w c o n c e n t r a t i o n s o f d i c u m a r o l . Hence t h e s e i n h i b i t o r s c o u l d a l s o b i n d t o c y t o c h r o m e P-450. D i c u m a r o l a t h i g h c o n c e n t r a -t i o n s e n h a n c e d t h e r a t e o f b r e a k d o w n o f c y t o c h r o m e P-450 t o c y t o c h r o m e P-420. Thus t h i s compound had two o p p o s i n g e f f e c t s o n c y t o c h r o m e P-4 50. The b i n d i n g o f d e o x y c o r t i c o s t e r o n e t o c y t o c h r o m e P-450 r e s u l t e d i n s p e c t r a l c h a n g e s i n t h e h e m o p r o t e i n t h a t c o u l d be m e a s u r e d by t h e t e c h n i q u e o f d i f f e r e n c e s p e c t r o p h o t o m e t r y . The s u b s t r a t e c o n c e n t r a t i o n r e q u i r e d f o r h a l f - m a x i m a l s p e c -t r a l c h a n g e , and h e n c e h a l f - m a x i m a l b i n d i n g t o c y t o c h r o m e P - 4 5 0 was a l m o s t i d e n t i c a l t o i t s K m f o r 1 1 3 - h y d r o x y l a t i o n . T h e d e o x y c o r t i c o s t e r o n e - i n d u c e d s p e c t r a l c h a n g e i n c y t o c h r o m e P - 4 5 0 was d i m i n i s h e d by a d d i t i o n o f M e t o p i r o n e o r b y h i g h c o n -c e n t r a t i o n s o f d i c u m a r o l b u t n o t b y low c o n c e n t r a t i o n s o f d i c u m a r o l . M e t o p i r o n e i n h i b i t s 1 1 3 - h y d r o x y l a t i o n by b i n d i n g t o c y t o c h r o m e P - 4 5 0 a n d p r e v e n t i n g t h e c o n c o m i t a n t b i n d i n g o f s u b s t r a t e d e o x y c o r t i c o s t e r o n e . T h e b i n d i n g o f M e t o p i r o n e a n d d e o x y c o r t i c o s t e r o n e t o c y t o c h r o m e P - 4 5 0 i s c o m p e t i t i v e i n n a t u r e , h e n c e c o m p e t i t i v e k i n e t i c s a r e o b s e r v e d w i t h M e t o p i r o n e i n h i b i t i o n o f t h e 1 1 3 - h y d r o x y l a t i o n r e a c t i o n . D i c u m a r o l e x e r t s two i n h i b i t o r y a c t i o n s o n 1 1 6 - h y d r o x y -l a t i o n . A t low c o n c e n t r a t i o n s t h i s compound b i n d s t o c y t o - . c h r o m e P - 4 5 0 b u t d o e s n o t a f f e c t s u b s t r a t e b i n d i n g t o "the<. hemo p r o t e i n , r e s u l t i n g i n n o n c o m p e t i t i v e i n h i b i t i o n o f 1 1 3 -h y d r o x y l a t i o n . T h e b i n d i n g o f d i c u m a r o l a t t h e s e c o n c e n t r a -t i o n s t h e r e f o r e m u s t i n h i b i t t h e i n t e r a c t i o n o f s t e r o i d s u b s t r a t e a n d o x y g e n t o d i m i n i s h t h e r a t e o f 1 1 3 - h y d r o x y l a t i o n D i c u m a r o l , a t h i g h c o n c e n t r a t i o n s , i n h i b i t s t h e b i n d i n g o f d e o x y c o r t i c o s t e r o n e t o c y t o c h r o m e P - 4 5 0 t h u s p r o d u c i n g i t s s e c o n d i n h i b i t o r y a c t i o n o n 1 1 3 - h y d r o x y l a t i o n . I n a d d i t i o n , b i n d i n g o f d i c u m a r o l a t h i g h c o n c e n t r a t i o n s r e s u l t s i n t h e b r e a k d o w n o f c y t o c h r o m e P - 4 5 0 . i v T A B L E OF CONTENTS P a g e A B S T R A C T i T A B L E OF CONTENTS i v L I S T OF T A B L E S v i i i L I S T OF F I G U R E S i x P U B L I C A T I O N S x i v A B B R E V I A T I O N S USED x v ACKNOWLEDGEMENTS x v i INTRODUCTION 1 S t e r o i d H y d r o x y l a s e s 2 S t e r o i d 1 1 3 - H y d r o x y l a s e 2 C o m p o n e n t s o f t h e 1 1 3 - H y d r o x y l a s e 6 NADPH R e d u c t a s e 8 Nonheme I r o n P r o t e i n 8 C y t o c h r o m e P - 4 5 0 9 C y t o c h r o m e P - 4 20 13 C y t o c h r o m e P - 4 5 0 a s t h e S u b s t r a t e B i n d i n g S i t e 14 M A T E R I A L S a n d METHODS 19 (a) S o l v e n t s 19 (b) C h e m i c a l s 19 (c) S t e r o i d s 20 (d) C h r o m a t o g r a p h y P r o c e d u r e s 20 (e) I s o l a t i o n o f t h e 1 1 3 - H y d r o x y l a s e f r o m B e e f A d r e n a l G l a n d s 21 ( i ) P r e p a r a t i o n o f b e e f a d r e n a l m i t o c h o n d r i a 21 ( i i ) P r e p a r a t i o n o f t h e m i t o c h o n d r i a l a c e t o n e powder 21 V P a g e ( i i i ) P r e p a r a t i o n o f t h e 1 1 3 - h y d r o x y l a s e f r o m s o n i c a t e d a d r e n a l m i t o c h o n d r i a 22 ( f ) P r o t e i n D e t e r m i n a t i o n s 23 (g) G e n e r a l I n c u b a t i o n C o n d i t i o n s 23 (h) S t u d i e s o n NADPH O x i d a t i o n 24 ( i ) S t u d i e s o n C o r t i c o s t e r o n e F o r m a t i o n ( 1 1 3 - H y d r o x y -l a t i o n ) 25 ( i ) K i n e t i c s t u d i e s o n 1 1 3 - h y d r o x y l a t i o n 26 ( i i ) I s o l a t i o n , p u r i f i c a t i o n , a n d m e a s u r e -ment o f d e o x y c o r t i c o s t e r o n e a n d c o r t i -c o s t e r o n e 26 ( i i i ) R a d i o a s s a y m e t h o d 29 ( j ) S p e c t r o p h o t o m e t r y A n a l y s i s o f t h e Enzyme P r e p a r a t i o n 29 (k) Ammonium S u l f a t e F r a c t i o n a t i o n o f t h e Enzyme P r e p a r a t i o n 30 (1) S t u d i e s o n C y t o c h r o m e s P - 4 5 0 a n d P - 4 2 0 31 ( i ) E f f e c t s o f 1 1 3 - h y d r o x y l a s e s u b s t r a t e s , i n h i b i t o r s ( D i c u m a r o l o r M e t o p i r o n e ) , a n d i n c u b a t i o n c o n d i t i o n s 31 ( i i ) S u b s t r a t e a n d i n h i b i t o r - i n d u c e d s p e c t r a l c h a n g e s i n c y t o c h r o m e P - 4 5 0 33 (m) S t u d i e s o n Bovine A d r e n a l M i t o c h o n d r i a l L i p i d s 35 ( i ) A c e t o n e - e x t r a c t e d l i p i d f r a c t i o n 35 ( i i ) P r e l i m i n a r y f r a c t i o n a t i o n o f t h e a c e t o n e -e x t r a c t e d l i p i d m a t e r i a l 35 ( i i i ) E x t r a c t i o n o f t h e t o t a l l i p i d s o f a d r e n a l m i t o c h o n d r i a 36 ( i v ) S i l i c a g e l c o l u m n c h r o m a t o g r a p h y o f t h e l i p i d f r a c t i o n s o l u b l e i n e t h a n o l a t - 2 0 ° C 38 (v) S a p o n i f i c a t i o n o f t h e a d r e n a l m i t o c h o n d r i a l a c e t o n e p o w d e r 39 v i P a g e PART I GENERAL P R O P E R T I E S OF THE l l g - H Y D R O X Y L A S E E X T R A C T E D FROM A B O V I N E ADRENAL MITOCHONDRIAL A C E T O N E POWDER 41 R E S U L T S (a) NADPH R e q u i r e m e n t o f t h e H B - H y d r o x y l a s e 41 (b) K i n e t i c S t u d y o f l l g - H y d r o x y l a t i o n 51 (c) S p e c t r o p h o t o m e t r i c S t u d i e s o n t h e Enzyme P r e p a r a -t i o n 53 ( i ) R e d u c e d - o x i d i z e d a n d c a r b o n m o n o x i d e d i f f e r e n c e s p e c t r a o f t h e enzyme p r e p a r a t i o n 55 ( i i ) Ammonium s u l f a t e f r a c t i o n a t i o n o f t h e enzyme p r e p a r a t i o n 59 (d) H B - H y d r o x y l a s e f r o m S o n i c a t e d A d r e n a l M i t o c h o n d r i a 65 ( i ) NADPH o x i d a t i o n a n d 1 1 3 - h y d r o x y l a t i o n by t h e s o n i c a t e d a d r e n a l m i t o c h o n d r i a p r e p a r a t i o n 65 ( i i ) E f f e c t o f a c e t o n e on t h e s o n i c a t e d enzyme p r e p a r a t i o n 71 (e) S t u d i e s o n C y t o c h r o m e P - 4 5 0 i n t h e A d r e n a l M i t o c h o n d r i a l A c e t o n e . Powder P r e p a r a t i o n 73 ( i ) E f f e c t o f i n c u b a t i o n t i m e a n d t e m p e r a t u r e o n c y t o c h r o m e s P - 4 5 0 a n d P - 4 2 0 74 ( i i ) E f f e c t o f s t e r o i d s u b s t r a t e s on c y t o -c h r o m e s P - 4 5 0 a n d P - 4 2 0 80 D I S C U S S I O N 87 PART I I THE ROLE OF QUINONES IN l l g - H Y D R O X Y L A T I O N 9 5 R E S U L T S 9 5 (a) E f f e c t o f A c e t o n e - E x t r a c t e d L i p i d a n d M e n a d i o n e ( V i t a m i n K 3 ) o n NADPH O x i d a t i o n 9 5 (b) E f f e c t o f A c e t o n e - E x t r a c t e d L i p i d a n d M e n a d i o n e o n 1 1 6 - H y d r o x y l a t i o n 101 v i i P a g e (c) A P o s s i b l e S i t e f o r M e n a d i o n e R e d u c t i o n 108 (d) E x a m i n a t i o n o f t h e A c e t o n e - E x t r a c t e d L i p i d F a c t o r s A f f e c t i n g NADPH O x i d a t i o n a n d l l g -H y d r o x y l a t i o n 113 (e) S t i m u l a t i o n o f l l g - H y d r o x y l a t i o n by t h e A c e t o n e -E x t r a c t e d L i p i d F r a c t i o n a n d b y A s o l e c t i n 122 D I S C U S S I O N 134 PART I I I DICUMAROL AND METOPIRONE I N H I B I T I O N OF l l g -HYDROXYLATION 142 R E S U L T S 14 2 (a) I n h i b i t i o n o f 1 1 6 - H y d r o x y l a t i o n by D i c u m a r o l 142 (b) I n h i b i t i o n o f H B - H y d r o x y l a t i o n b y M e t o p i r o n e 149 D I S C U S S I O N PART I V E F F E C T OF DICUMAROL AND METOPIRONE ON C Y T O -CHROMES P - 4 5 0 AND P - 4 2 0 164 R E S U L T S 164 (a) E f f e c t o f D i c u m a r o l o n C y t o c h r o m e s P - 4 5 0 a n d P - 4 2 0 164 (b) E f f e c t o f M e t o p i r o n e o n C y t o c h r o m e s P - 4 5 0 a n d P - 4 2 0 179 (c) S u b s t r a t e a n d I n h i b i t o r - I n d u c e d D i f f e r e n c e S p e c t r a o f C y t o c h r o m e P - 4 5 0 189 ( i ) S u b s t r a t e - i n d u c e d d i f f e r e n c e s p e c t r u m o f c y t o c h r o m e P - 4 5 0 191 ( i i ) E f f e c t o f d i c u m a r o l o n t h e s u b s t r a t e -i n d u c e d d i f f e r e n c e s p e c t r u m o f c y t o -c h r o m e P - 4 5 0 . 193 ( i i i ) M e t o p i r o n e - i n d u c e d d i f f e r e n c e s p e c t r u m o f c y t o c h r o m e P - 4 5 0 200 ( i v ) E f f e c t o f M e t o p i r o n e o n t h e s u b s t r a t e -i n d u c e d d i f f e r e n c e s p e c t r u m o f c y t o -c h r o m e P - 4 5 0 202 D I S C U S S I O N 205 B I B L I O G R A P H Y 222 L I S T OF T A B L E S v i i i T a b l e P a g e I E f f e c t o f i n c r e a s i n g q u a n t i t i e s o f NADPH o n t h e 1 1 6 - h y d r o x y l a t i o n o f d e o x y c o r t i c o s t e r o n e 42 I I 1 1 6 - h y d r o x y l a t i o n o f d e o x y c o r t i c o s t e r o n e as a f u n c t i o n o f t i m e 46 I I I E f f e c t o f i n h i b i t o r s o n NADPH o x i d a t i o n 48 I V E f f e c t o f i n h i b i t o r s o n t h e 1 1 6 - h y d r o x y l a t i o n o f d e o x y c o r t i c o s t e r o n e 50 V C o m p a r i s o n o f t h e 116 - h y d r o x y l a s e a c t i v i t y a n d c y t o c h r o m e P-4 50 c o n t e n t i n t h e enzyme s y s t e m s p r e p a r e d b y e x t r a c t i o n o f a n a d r e n a l m i t o c h o n d r i a l a c e t o n e p o w d e r a n d by u l t r a c e n t r i f u g a t i o n o f s o n i -c a t e d a d r e n a l m i t o c h o n d r i a 70 V I R e c o v e r y o f c y t o c h r o m e s P-450 a n d P-420 f r o m t h e m i t o c h o n d r i a l a c e t o n e p o w d e r enzyme p r e p a r a t i o n i n c u b a t e d i n t h e p r e s e n c e o r a b s e n c e o f NADPH 79 V I I E f f e c t o f m i t o c h o n d r i a l a c e t o n e - e x t r a c t e d l i p i d , m e n a d i o n e , a n d d i c u m a r o l o n t h e i n i t i a l r a t e o f NADPH o x i d a t i o n 102 V I I I T h e r e l a t i o n s h i p b e t w e e n o x i d a t i o n o f NADPH a n d r e d u c t i o n o f c y t o c h r o m e s P-450 a n d P-420 i n t h e 20-40% a n d 40-80% ammonium s u l f a t e f r a c t i o n s o f t h e m i t o c h o n d r i a l a c e t o n e powder enzyme p r e p a r a -t i o n 110 I X E f f e c t o f a d r e n a l m i t o c h o n d r i a l i p i d f r a c t i o n s o n t h e 1 1 6 - h y d r o x y l a t i o n o f d e o x y c o r t i c o s t e r o n e a n d o n NADPH o x i d a t i o n 115 X E f f e c t o f a d r e n a l m i t o c h o n d r i a l i p i d f r a c t i o n s o n NADPH o x i d a t i o n 118 X I E f f e c t o f s i l i c a g e l c o l u m n e l u a t e s o n NADPH o x i d a t i o n 120 X I I E f f e c t o f d i c u m a r o l o n t h e 113 - h y d r o x y l a s e a c t i v i t y , c y t o c h r o m e P-450, a n d c y t o c h r o m e P-420 c o n t e n t o f t h e enzyme s y s t e m p r e p a r e d by s o n i c a t i o n o f a d r e n a l m i t o c h o n d r i a 178 X I I I E f f e c t o f M e t o p i r o n e o n c y t o c h r o m e s P-4 50 a n d P-420 i n t h e enzyme s y s t e m p r e p a r e d b y s o n i c a t i o n o f a d r e n a l m i t o c h o n d r i a 190 IX L I S T OF F I G U R E S F i g u r e P a g e 1. Scheme p r o p o s e d b y Omura et at. f o r t h e e l e c t r o n t r a n s p o r t p a t h w a y i n h y d r o x y l a t i o n r e a c t i o n s 7 2 . T h e t r a n s f o r m a t i o n o f d e o x y c o r t i c o s t e r o n e t o c o r t i -c o s t e r o n e 18 3. S t r u c t u r a l f o r m u l a e o f d i c u m a r o l a n d M e t o p i r o n e 18 4. O x i d a t i o n o f NADPH i n t h e p r e s e n c e a n d a b s e n c e o f d e o x y c o r t i c o s t e r o n e 45 5 . T h e r a t e o f 1 1 3 - h y d r o x y l a t i o n as a f u n c t i o n o f d e o x y c o r t i c o s t e r o n e c o n c e n t r a t i o n 52 6. [ S ] / v v e r s u s [S] p l o t f o r t h e 1 1 3 - h y d r o x y l a t i o n o f d e o x y c o r t i c o s t e r o n e 54 7 . R e d u c e d m i n u s o x i d i z e d d i f f e r e n c e s p e c t r u m o f t h e m i t o c h o n d r i a l a c e t o n e p o w d e r enzyme p r e p a r a t i o n r e d u c e d w i t h NADPH o r s o d i u m d i t h i o n i t e 56 8. C a r b o n m o n o x i d e d i f f e r e n c e s p e c t r u m o f t h e m i t o c h o n -d r i a l a c e t o n e p o w d e r enzyme p r e p a r a t i o n r e d u c e d w i t h NADPH o r s o d i u m d i t h i o n i t e 58 9 a . D i t h i o n i t e r e d u c e d m i n u s o x i d i z e d d i f f e r e n c e s p e c t r u m o f t h e 0-20% ammonium s u l f a t e f r a c t i o n o f t h e m i t o -c h o n d r i a l a c e t o n e powder enzyme p r e p a r a t i o n 60 9 b . D i t h i o n i t e r e d u c e d c a r b o n m o n o x i d e d i f f e r e n c e s p e c t r u m o f t h e 0-20% ammonium s u l f a t e f r a c t i o n o f t h e m i t o -c h o n d r i a l a c e t o n e powder enzyme p r e p a r a t i o n 60 1 0 a . D i t h i o n i t e r e d u c e d m i n u s o x i d i z e d d i f f e r e n c e s p e c t r u m o f t h e 20-40% ammonium s u l f a t e f r a c t i o n o f t h e m i t o -c h o n d r i a l a c e t o n e p o w d e r enzyme p r e p a r a t i o n 61 1 0 b . D i t h i o n i t e r e d u c e d c a r b o n m o n o x i d e d i f f e r e n c e s p e c t r u m o f t h e 20-40% ammonium s u l f a t e f r a c t i o n o f t h e m i t o -c h o n d r i a l a c e t o n e powder enzyme p r e p a r a t i o n 61 1 1 a . D i t h i o n i t e r e d u c e d m i n u s o x i d i z e d d i f f e r e n c e s p e c t r u m o f t h e 40-80% ammonium s u l f a t e f r a c t i o n o f t h e m i t o -c h o n d r i a l a c e t o n e p o w d e r enzyme p r e p a r a t i o n 6 2 l i b . D i t h i o n i t e r e d u c e d c a r b o n m o n o x i d e d i f f e r e n c e s p e c t r u m o f t h e 40-80% ammonium s u l f a t e f r a c t i o n o f t h e m i t o -c h o n d r i a l a c e t o n e p o w d e r enzyme p r e p a r a t i o n 62 1 2 . NADPH o x i d a t i o n b y a n enzyme p r e p a r a t i o n o b t a i n e d b y u l t r a c e n t r i f u g a t i o n o f s o n i c a t e d a d r e n a l m i t o c h o n d r i a 66 X F i g u r e P a g e 1 3 . NADPH r e d u c e d c a r b o n m o n o x i d e d i f f e r e n c e s p e c t r u m o f a n enzyme p r e p a r a t i o n o b t a i n e d by u l t r a c e n t r i -f u g a t i o n o f s o n i c a t e d a d r e n a l m i t o c h o n d r i a 69 1 4 . E f f e c t o f e x t r a c t i o n w i t h a c e t o n e o r a c e t o n e - w a t e r ( 9 : 1 v / v ) o n t h e c y t o c h r o m e P - 4 5 0 c o n t e n t o f t h e s o n i c a t e d a d r e n a l m i t o c h o n d r i a enzyme p r e p a r a t i o n 72 1 5 . E f f e c t o f i n c u b a t i o n t e m p e r a t u r e o n t h e r e c o v e r y o f c y t o c h r o m e s P - 4 5 0 a n d P - 4 2 0 f r o m t h e a d r e n a l m i t o -c h o n d r i a l a c e t o n e p o w d e r enzyme p r e p a r a t i o n 75 1 6 . C o n v e r s i o n o f c y t o c h r o m e P - 4 5 0 t o c y t o c h r o m e P - 4 2 0 a s a f u n c t i o n o f i n c u b a t i o n t i m e 77 1 7 . R e c o v e r y o f c y t o c h r o m e P - 4 5 0 f r o m t h e m i t o c h o n d r i a l a c e t o n e p o w d e r enzyme p r e p a r a t i o n a f t e r i n c u b a t i o n i n t h e p r e s e n c e o f d e o x y c o r t i c o s t e r o n e o r c o r t i c o -s t e r o n e 82 1 8 . R e c o v e r y o f c y t o c h r o m e P - 4 5 0 f r o m t h e m i t o c h o n d r i a l a c e t o n e powder enzyme p r e p a r a t i o n a f t e r i n c u b a t i o n i n t h e p r e s e n c e o f a n d r o s t e n e d i o n e o r a d r e n o s t e r o n e 83 1 9 . R e c o v e r y o f c y t o c h r o m e s P - 4 5 0 a n d P - 4 2 0 f r o m t h e m i t o c h o n d r i a l a c e t o n e powder enzyme p r e p a r a t i o n a f t e r i n c u b a t i o n i h r t h e p r e s e n c e o f h i g h c o n c e n t r a t i o n s o f d e o x y c o r t i c o s t e r o n e 8 5 2 0 . E f f e c t o f d e o x y c o r t i c o s t e r o n e o n t h e r a t e o f c y t o -c h r o m e P - 4 5 0 d i s a p p e a r a n c e 86 2 1 . E f f e c t o f t h e a c e t o n e - e x t r a c t e d m i t o c h o n d r i a l l i p i d f r a c t i o n o n NADPH o x i d a t i o n 97 2 2 . E f f e c t o f m e n a d i o n e o n NADPH o x i d a t i o n 98 2 3 . E f f e c t o f 1 , 4 - n a p h t h o q u i n o n e , 1 , 4 - t o l u o q u i n o n e , a n d 1 , 4 - b e n z o q i i i n o n e o n NADPH o x i d a t i o n 10 0 2 4 . E f f e c t o f d e o x y c o r t i c o s t e r o n e , m i t o c h o n d r i a l a c e t o n e -e x t r a c t e d l i p i d , a n d d i c u m a r o l on NADPH o x i d a t i o n 10 4 2 5 . E f f e c t o f d e o x y c o r t i c o s t e r o n e , m e n a d i o n e , a n d d i -c u m a r o l o n NADPH o x i d a t i o n 106 2 6 . E f f e c t o f m i t o c h o n d r i a l a c e t o n e - e x t r a c t e d l i p i d , m e n a d i o n e , a n d d i c u m a r o l o n t h e 1 1 6 - h y d r o x y l a t i o n o f d e o x y c o r t i c o s t e r o n e 107 XI F i g u r e P a g e 2 7 a . E f f e c t o f m e n a d i o n e o n t h e o x i d a t i o n o f NADPH b y t h e 20-40% ammonium s u l f a t e f r a c t i o n o f t h e m i t o c h o n d r i a l a c e t o n e powder enzyme p r e p a r a t i o n 112 2 7 b . E f f e c t o f m e n a d i o n e o n t h e NADPH m e d i a t e d r e d u c t i o n o f c y t o c h r o m e s P - 4 5 0 a n d P - 4 2 0 i n t h e 20-40% ammonium s u l f a t e f r a c t i o n o f t h e m i t o c h o n d r i a l a c e t o n e p o w d e r enzyme p r e p a r a t i o n 112 2 8 . E f f e c t o f m i t o c h o n d r i a l l i p i d a n d a s o l e c t i n o n t h e 1 1 3 - h y d r o x y l a t i o n o f d e o x y c o r t i c o s t e r o n e 124 2 9 . 1 1 3 - h y d r o x y l a t i o n o f d e o x y c o r t i c o s t e r o n e a s a f u n c t i o n o f m i t o c h o n d r i a l l i p i d o r a s o l e c t i n c o n -c e n t r a t i o n 125 3 0 . E f f e c t o f d e o x y c o r t i c o s t e r o n e a n d m i t o c h o n d r i a l l i p i d o n c y t o c h r o m e P - 4 5 0 127 3 1 . E f f e c t o f t o t a l m i t o c h o n d r i a l l i p i d a n d - 2 0 ° C a c e t o n e - i n s o l u b l e f r a c t i o n o n c y t o c h r o m e s P - 4 5 0 a n d P - 4 2 0 128 3 2 . R e c o v e r y o f c y t o c h r o m e s P - 4 5 0 a n d P - 4 2 0 as a f u n c -t i o n o f t h e m i t o c h o n d r i a l l i p i d c o n c e n t r a t i o n 130 3 3 . R e c o v e r y o f c y t o c h r o m e s P - 4 5 0 a n d P - 4 2 0 as a f u n c -t i o n o f a s o l e c t i n c o n c e n t r a t i o n 131 3 4 . E f f e c t o f m i t o c h o n d r i a l l i p i d o n t h e r a t e o f c y t o -c h r o m e P - 4 5 0 d i s a p p e a r a n c e 133 3 5 . S c h e m a t i c r e p r e s e n t a t i o n o f t h e i n t e r a c t i o n o f m e n a d i o n e w i t h t h e c o m p o n e n t s o f t h e e l e c t r o n t r a n s p o r t p a t h w a y f o r 1 1 3 - h y d r o x y l a t i o n 137 3 6 a . E f f e c t o f d i c u m a r o l a n d d e o x y c o r t i c o s t e r o n e o n NADPH o x i d a t i o n 14 3 3 6 b . E f f e c t o f d i c u m a r o l o n t h e 1 1 3 - h y d r o x y l a t i o n o f d e o x y c o r t i c o s t e r o n e 143 3 7 . I n h i b i t i o n o f t h e 1 1 3 - h y d r o x y l a t i o n o f d e o x y c o r t i -c o s t e r o n e as a f u n c t i o n o f d i c u m a r o l c o n c e n t r a t i o n 145 3 8 . [ S ] / v v e r s u s [S] p l o t o f d i c u m a r o l i n h i b i t i o n o f t h e 1 1 3 - h y d r o x y l a t i o n o f d e o x y c o r t i c o s t e r o n e 146 3 9 . 1 / v v e r s u s i p l o t o f d i c u m a r o l i n h i b i t i o n o f t h e 1 1 3 - h y d r o x y l a t i o n o f d e o x y c o r t i c o s t e r o n e 148 x i i F i g u r e P a g e 4 0 . NADPH o x i d a t i o n as a f u n c t i o n o f M e t o p i r o n e c o n c e n -t r a t i o n 150 4 1 . I n h i b i t i o n o f t h e 1 1 6 - h y d r o x y l a t i o n o f d e o x y c o r t i -c o s t e r o n e a s a f u n c t i o n o f M e t o p i r o n e c o n c e n t r a t i o n 152 4 2 a . E f f e c t o f M e t o p i r o n e a n d d e o x y c o r t i c o s t e r o n e on NADPH= o x i d a t i o n 154 4 2 b . E f f e c t o f M e t o p i r o n e o n t h e 1 1 6 - h y d r o x y l a t i o n o f d e o x y c o r t i c o s t e r o n e 154 4 3 . 1 / v v e r s u s i p l o t o f M e t o p i r o n e i n h i b i t i o n o f 1 1 6 -h y d r o x y l a t i o n o f d e o x y c o r t i c o s t e r o n e 155 4 4 . R e c o v e r y o f c y t o c h r o m e s P - 4 5 0 a n d P - 4 2 0 f r o m t h e m i t o c h o n d r i a l a c e t o n e p o w d e r enzyme p r e p a r a t i o n a f t e r i n c u b a t i o n i n t h e p r e s e n c e o f d i c u m a r o l 166 4 5 . R e c o v e r y o f c y t o c h r o m e s P - 4 5 0 a n d P - 4 2 0 f r o m t h e m i t o c h o n d r i a l a c e t o n e p o w d e r - e n z y m e p r e p a r a t i o n a f t e r i n c u b a t i o n i n t h e p r e s e n c e o f l ow c o n c e n -t r a t i o n s o f d i c u m a r o l 167 4 6 . R e c o v e r y o f c y t o c h r o m e s P - 4 5 0 a n d P - 4 2 0 f r o m t h e m i t o c h o n d r i a l a c e t o n e powder enzyme p r e p a r a t i o n a f t e r i n c u b a t i o n i n t h e p r e s e n c e o f h i g h c o n c e n -t r a t i o n s o f d i c u m a r o l 169 4 7 . R e c o v e r y o f c y t o c h r o m e P - 4 5 0 f r o m t h e m i t o c h o n d r i a l a c e t o n e powder enzyme p r e p a r a t i o n a f t e r i n c u b a t i o n i n t h e p r e s e n c e o f d i c u m a r o l a n d d e o x y c o r t i c o s t e r o n e 170 4 8 . E f f e c t o f d e o x y c o r t i c o s t e r o n e a n d d i c u m a r o l o n t h e r a t e o f c y t o c h r o m e P - 4 5 0 d i s a p p e a r a n c e 174 4 9 . E f f e c t o f d i c u m a r o l on c y t o c h r o m e P - 4 5 0 i n t h e 1 1 6 - h y d r o x y l a s e p r e p a r e d f r o m s o n i c a t e d a d r e n a l m i t o c h o n d r i a 176 5 0 . R e c o v e r y o f c y t o c h r o m e s P - 4 5 0 a n d P - 4 2 0 f r o m t h e m i t o c h o n d r i a l a c e t o n e p o w d e r enzyme p r e p a r a t i o n a f t e r i n c u b a t i o n i n t h e p r e s e n c e o f M e t o p i r o n e 181 5 1 . E f f e c t o f M e t o p i r o n e o n t h e r a t e o f c y t o c h r o m e P - 4 5 0 d i s a p p e a r a n c e 182 5 2 . R e c o v e r y o f c y t o c h r o m e s P - 4 5 0 a n d P - 4 2 0 f r o m t h e m i t o c h o n d r i a l a c e t o n e powder enzyme p r e p a r a t i o n a f t e r i n c u b a t i o n i n t h e p r e s e n c e o f l ow c o n c e n t r a -t i o n s o f M e t o p i r o n e 18 5 x i i i F i g u r e , Page 5 3 . R e c o v e r y o f c y t o c h r o m e P - 4 5 0 f r o m t h e m i t o c h o n d r i a l a c e t o n e p o w d e r enzyme p r e p a r a t i o n a f t e r i n c u b a t i o n i n t h e p r e s e n c e o f M e t o p i r o n e a n d NADPH 18 6 5 4 . R e c o v e r y o f c y t o c h r o m e P - 4 5 0 f r o m t h e m i t o c h o n d r i a l a c e t o n e p o w d e r enzyme p r e p a r a t i o n a f t e r i n c u b a t i o n i n t h e p r e s e n c e o f M e t o p i r o n e a n d d e o x y c o r t i c o s t e r o n e 188 5 5 . S p e c t r a l c h a n g e s p r o d u c e d b y a d d i t i o n o f d e o x y c o r t i -c o s t e r o n e t o t h e 20-40% ammonium s u l f a t e f r a c t i o n 192 o f t h e m i t o c h o n d r i a l a c e t o n e powder enzyme p r e p a r a t i o n 5 6 . T h e e f f e c t o f d e o x y c o r t i c o s t e r o n e c o n c e n t r a t i o n o n t h e m a g n i t u d e o f t h e s p e c t r a l c h a n g e p r o d u c e d b y a d -d i t i o n o f d e o x y c o r t i c o s t e r o n e t o t h e 20-40% ammonium s u l f a t e f r a c t i o n o f t h e m i t o c h o n d r i a l a c e t o n e powder enzyme p r e p a r a t i o n 19 4 5 7 . D o u b l e r e c i p r o c a l p l o t o f t h e c h a n g e i n a b s o r b a n c e ( 385 -420 my) o r ( 420 -403 my) a s a f u n c t i o n o f d e o x y -c o r t i c o s t e r o n e c o n c e n t r a t i o n 195 5 8 . E f f e c t o f d i c u m a r o l o n t h e s p e c t r a l c h a n g e s p r o d u c e d by a d d i t i o n o f d e o x y c o r t i c o s t e r o n e t o t h e 20-40% ammonium s u l f a t e f r a c t i o n o f t h e m i t o c h o n d r i a l a c e t o n e p o w d e r enzyme p r e p a r a t i o n 19 7 5 9 . E f f e c t o f d i c u m a r o l o n t h e m a g n i t u d e o f t h e s p e c -t r a l c h a n g e ( 3 8 5 - 4 2 0 my) p r o d u c e d b y a d d i t i o n o f d e o x y c o r t i c o s t e r o n e t o t h e 20-40% ammonium s u l f a t e f r a c t i o n o f t h e m i t o c h o n d r i a l a c e t o n e p o w d e r enzyme p r e p a r a t i o n 198 6 0 . S p e c t r a l c h a n g e s p r o d u c e d by a d d i t i o n o f M e t o p i r o n e t o t h e 20-40% ammonium s u l f a t e f r a c t i o n o f t h e m i t o c h o n d r i a l a c e t o n e p o w d e r enzyme p r e p a r a t i o n 201 6 1 . E f f e c t o f M e t o p i r o n e o n t h e s p e c t r a l c h a n g e s p r o -d u c e d by a d d i t i o n o f d e o x y c o r t i c o s t e r o n e t o t h e 20-40% ammonium s u l f a t e f r a c t i o n o f t h e m i t o c h o n -d r i a l a c e t o n e powder enzyme p r e p a r a t i o n 2 03 6 2 . E f f e c t o f M e t o p i r o n e o n t h e m a g n i t u d e o f t h e s p e c t r a l c h a n g e (385-420 my) p r o d u c e d b y a d d i t i o n o f d e o x y c o r t i c o s t e r o n e t o t h e 20-40% ammonium s u l f a t e f r a c t i o n o f t h e m i t o c h o n d r i a l a c e t o n e p o w d e r enzyme p r e p a r a t i o n 20 4 6 3 . S c h e m a t i c r e p r e s e n t a t i o n o f t h e c o n v e r s i o n o f c y t o -c h r o m e P - 4 5 0 t o d i f f e r e n t c o n f o r m a t i o n a l s t a t e s b y i n t e r a c t i o n w i t h d e o x y c o r t i c o s t e r o n e , M e t o p i r o n e , a n d d i c u m a r o l 217 x i v P U B L I C A T I O N S W i l l i a m s o n , D . a n d O ' D o n n e l l , V . J . T h e E n z y m a t i c M e c h a n i s m o f 1 1 6 - H y d r o x y l a t i o n o f C o r t i c o s t e r o i d s . P r o c . C a n . F e d . B i o l . S o c . , 9 , 51 ( 1 9 6 6 ) . W i l l i a m s o n , D . G . , a n d O ' D o n n e l l , V . J . M e c h a n i s m o f M e t o p i r o n e I n h i b i t i o n o f a S o l u b l e A d r e n a l S t e r o i d 1 1 6 - H y d r o x y l a s e . C a n . J . B i o c h e m . , _45, 153 ( 1 9 6 7 ) . W i l l i a m s o n , D . G . a n d O ' D o n n e l l , V . J . D i c u m a r o l : A N o v e l I n h i b i t o r o f S t e r o i d 1 1 6 - H y d r o x y l a t i o n . C a n . J . B i o c h e m . , 4 5 , 340 ( 1 9 6 7 ) . W i l l i a m s o n , D . a n d O ' D o n n e l l , V . J . D i c u m a r o l I n h i b i t i o n o f S t e r o i d 1 1 6 - H y d r o x y l a t i o n . P r o c . C a n . F e d . B i o l . S o c , 1 0 , 64 ( 1 9 6 7 ) . XV ABBREVIATIONS USED D e o x y c o r t i c o s t e r o n e : 2 1 - h y d r o x y p r e g n - 4 - e n e - 3 , 2 0 - d i o n e C o r t i c o s t e r o n e : 1 1 3 , 2 1 - d i h y d r o x y p r e g n - 4 - e n e - 3 , 2 0 - d i o n e D e o x y c o r t i s o l : 1 7 , 2 1 - d i h y d r o x y p r e g n - 4 - e n e - 3 , 2 0 - d i o n e A n d r o s t e n e d i o n e : a n d r o s t - 4 - e n e - 3 , 1 7 - d i o n e T e s t o s t e r o n e : 1 7 3 - h y d r o x y a n d r o s t - 4 - e n - 3 - o n e 1 1 3 - H y d r o x y a n d r o s t e n e d i o n e : 1 1 3 - h y d r o x y a n d r o s t - 4 - e n e - 3 , 1 7 - d i o n e A d r e n o s t e r o n e : a n d r o s t - 4 - e n e - 3 , 1 1 , 1 7 - t r i o n e D e h y d r o e p i a n d r o s t e r o n e : 3 3 - h y d r o x y a n d r o s t - 5 - e n - 1 7 - o n e D i c u m a r o l : 3 , 3 ' - m e t h y l e n e b i s - ( 4 - h y d r o x y c o u m a r i n ) M e t o p i r o n e : 2 - m e t h y l - l , 2 - b i s ( 3 - p y r i d y l ) - 1 - p r o p a n o n e M e n a d i o n e : 2 - m e t h y l - l , 4 - n a p h t h o q u i n o n e Coenzyme Q: 2 , 3 - d i m e t h o x y - 5 - m e t h y l - 6 - m u l t i p r e n y l - l , 4 - b e n z o q u i n o n e NAD: o x i d i z e d 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 NADH:- r e d u c e d NAD-NADP: o x i d i z e d 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 p h o s p h a t e NADPH: r e d u c e d NADP ATP: a d e n o s i n e t r i p h o s p h a t e PPO: 2 , 5 - d i p h e n y l o x a z o l e POPOP: 1 , 4 - b i s [ 2 - ( 5 - p h e n y l o x a z o l y l ) ] b e n z e n e x v i ACKNOWLEDGEMENTS I w i s h t o e x p r e s s my d e e p e s t t h a n k s t o D r . V . J . O ' D o n n e l l f o r h i s g u i d a n c e and e n c o u r a g e m e n t t h r o u g h o u t t h e s e s t u d i e s a n d f o r h i s i n v a l u a b l e a s s i s t a n c e i n t h e p r e p a r a t i o n o f t h i s t h e s i s . I w o u l d a l s o l i k e t o t h a n k D r . P.D. B r a g g f o r h i s a d v i c e on t h e e x p e r i m e n t s w i t h l i p o q u i n o n e s a n d on t h e t e c h n i q u e o f d i f -f e r e n c e s p e c t r o p h o t o m e t r y . I w o u l d l i k e t o a c k n o w l e d g e t h e N a t i o n 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 a n d t h e H.R. M a c M i l l a n F a m i l y f o r f i n a n c i a l s u p p o r t i n t h e f o r m o f s c h o l a r s h i p s a w a r d e d t o me d u r i n g t h e s e s t u d i e s . The r e s e a r c h was s u p p o r t e d b y g r a n t s t o D r . V . J . O ' D o n n e l l f r o m t h e 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 a n d f r o m t h e N a t i o n a l C a n c e r I n s t i t u t e o f C a n a d a . - 1 -INTRODUCTION I n 19 55 M a s o n, F o w l k s , a n d P e t e r s o n o b s e r v e d (1) t h a t d u r i n g t h e e n z y m a t i c o x i d a t i o n o f 3 , 4 - d i m e t h y l p h e n o l t o 4,5-d i m e t h y l c a t e c h o l t h e o x y g e n atom i n c o r p o r a t e d i n t o t h e s u b -s t r a t e m o l e c u l e was d e r i v e d e x c l u s i v e l y f r o m m o l e c u l a r o x y g e n . T h i s o b s e r v a t i o n h a s l e d t o t h e d i s c o v e r y o f a number o f e n -zymes c a t a l y z i n g s i m i l a r r e a c t i o n s ( 2 , 3 ) . H a y a i s h i (2) i n t r o -d u c e d t h e t e r m " o x y g e n a s e " t o d e s c r i b e enzymes o f t h i s t y p e . T h e s e enzymes i n g e n e r a l c a t a l y z e t h e i n t r o d u c t i o n o f one o r two atoms o f m o l e c u l a r o x y g e n i n t o t h e s u b s t r a t e m o l e c u l e . I n o x i d a t i o n r e a c t i o n s i n w h i c h o n l y one atom o f m o l e c u l a r o x y g e n i s i n c o r p o r a t e d i n t o t h e s u b s t r a t e , t h e s e c o n d atom i s r e d u c e d t o w a t e r i n t h e p r e s e n c e o f a n a p p r o p r i a t e e l e c t r o n d o n o r s u c h a s NADH, NADPH, t e t r a h y d r o f o l a t e , o r a s c o r b i c a c i d ( 2 - 4 ) . Mason (3) i n t r o d u c e d t h e t e r m " m i x e d f u n c t i o n o x i d a s e " t o d e s c r i b e enzymes c a t a l y z i n g t h i s p a r t i c u l a r t y p e o f r e a c t i o n b e c a u s e t h e y p o s s e s s b o t h o x y g e n a s e a c t i v i t y ( i n c o r p o r a t i o n o f an atom o f m o l e c u l a r o x y g e n i n t o t h e s u b s t r a t e ) , a n d o x i d a s e a c t i v i t y ( r e d u c t i o n o f a n atom o f m o l e c u l a r o x y g e n t o w a t e r ) . The t e r m s "monooxygenase" (5) a n d " h y d r o x y l a s e " (6) h a v e a l s o b e e n e m p l o y e d t o d e s c r i b e t h e s e e nzymes. The p r o p e r t i e s and r e q u i r e m e n t s o f t h e e n z y m e s . c a t a l y z i n g h y d r o x y l a t i o n s o f s t e r o i d hormones p l a c e them i n t h e g r o u p o f m i x e d f u n c t i o n o x i d a s e s . The r e q u i r e m e n t s o f t h e m i x e d f u n c t i o n o x i d a s e s a r e u n -u s u a l i n t h a t o x y g e n i s r e q u i r e d a s a s p e c i f i c o x i d i z i n g a g e n t - 2 -a n d an e l e c t r o n d o n o r i s n e e d e d a s a r e d u c t a n t . I n s t e r o i d h y d r o x y l a s e s NADPH i s u s u a l l y t h e r e d u c t a n t (7,8,9) w h i l e some o t h e r t y p e s o f h y d r o x y l a s e s a r e s p e c i f i c f o r NADH ( 1 0 , 1 1 ) . A s c o r b i c a c i d s e r v e s as r e d u c t a n t i n d o p a m i n e h y d r o x y -l a s e (12) a n d t e t r a h y d r o p t e r i d i n e i s r e q u i r e d i n t h e p h e n y l -a l a n i n e h y d r o x y l a s e s y s t e m ( 1 3 ) . S t e r o i d H y d r o x y l a s e s The s t e r o i d m o l e c u l e i s h y d r o x y l a t e d a t a number o f p o s i t i o n s i n b o t h a n i m a l t i s s u e s (14) and b a c t e r i a ( 1 5 - 1 8 ) . M a j o r e f f o r t s i n t h e s t u d y o f t h e e n z y m a t i c m e c h a n i s m o f mammalian s t e r o i d h y d r o x y l a t i o n s h a v e b e e n d i r e c t e d t o w a r d t h e 1 1 3 - , 1 7 a - , and 2 1 - h y d r o x y l a s e s o f t h e a d r e n a l c o r t e x ( 1 4 , 1 9 ) . Of t h e s e , t h e 1 1 3 - h y d r o x y l a s e h a s b e e n s t u d i e d m o s t e x t e n s i v e l y a n d i s t h e s u b j e c t o f t h i s t h e s i s . The 1 1 3 - h y d r o x y l f u n c t i o n o f c o r t i c o s t e r o i d s h a s h a d p a r t i c u l a r s i g n i f i c a n c e a t t a c h e d t o i t i n t e r m s o f hormone a c t i v i t y b y B u s h a n d Mahesh (20) . They s u g g e s t t h a t t h e b i o l o g i c a l a c t i v i t y o f 1 1 -o x y g e n a t e d s t e r o i d s a s g l u c o c o r t i c o i d s may be due t o t h e s p e -c i f i c i n t e r a c t i o n o f a n 1 1 3 - h y d r o x y l g r o u p w i t h t h e r e c e p t o r s i t e f o r t h e s e h o r m o n e s . S t e r o i d 1 1 3 - H y d r o x y l a s e The s t e r o i d 1 1 3 - h y d r o x y l a s e i s l o c a t e d 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 o f a d r e n o c o r t i c a l t i s s u e ( 2 1 - 2 5 ) . A l s o p r e s e n t i n t h i s s u b c e l l u l a r f r a c t i o n a r e t h e 1 8 - h y d r o x y l a s e (26) and h y d r o x y l a s e s i n v o l v e d i n t h e s i d e - c h a i n c l e a v a g e o f c h o l e s t e r o l t o p r e g n e n o l o n e ( 2 7 ) . The 1 7 a - and 2 1 - h y d r o x y l a s e s o n t h e - 3 -o t h e r h a n d a r e a s s o c i a t e d w i t h t h e e n d o p l a s m i c r e t i c u l u m (28 -30) . Th e 1 1 3 - h y d r o x y l a s e o f t h e a d r e n a l c o r t e x h a s b e e n p r e -p a r e d by a v a r i e t y o f m e t h o d s . T h e s y s t e m c a n be e x t r a c t e d f r o m a n a c e t o n e p o w d e r o f a d r e n a l m i t o c h o n d r i a (31) a n d p a r -t i a l l y e x t r a c t e d f r o m l y o p h i l i z e d m i t c h o n d r i a ( 3 2 ) . S h a r m a et at. (33) h a v e o b t a i n e d a n a c t i v e p r e p a r a t i o n f r o m b o v i n e a d r e n a l c o r t e x by u l t r a s o n i c t r e a t m e n t o f a d r e n a l m i t o c h o n d r i a f o l l o w e d by c e n t r i f u g a t i o n . T h e p r o p e r t i e s o f t h e 1 1 3 - h y d r o x y -l a s e f r o m t h e s e v a r i o u s p r e p a r a t i o n s h a v e b e e n e x a m i n e d i n d e t a i l . I n e a r l y s t u d i e s o n t h e c o f a c t o r r e q u i r e m e n t s o f t h e 113-h y d r o x y l a s e a number o f c o m p o n e n t s a p p e a r e d e s s e n t i a l . A n o x y g e n - c o n t a i n i n g a t m o s p h e r e was r e q u i r e d f o r h y d r o x y l a t i o n ( 2 2 , 2 4 , 2 5 , 3 4 ) . T h e r e a c t i o n a l s o a p p e a r e d t o be d e p e n d e n t o n , o r s t i m u l a t e d b y a v a r i e t y o f f a c t o r s , i n c l u d i n g , A T P , M g + + , N A D , NADP, n i c o t i n a m i d e a n d f u m a r a t e o r o t h e r K r e b s c y c l e i n -t e r m e d i a t e s ( 2 2 - 2 5 ) . I n a d d i t i o n , B r o w n i e a n d G r a n t (24) n o t e d t h a t s u c c i n a t e - s u p p o r t e d 1 1 3 - h y d r o x y l a t i o n was i n h i b i t e d b y d i n i t r o p h e n y l , s u g g e s t i n g t h a t o x i d a t i v e p h o s p h o r y l a t i o n was n e c e s s a r y f o r 1 1 3 - h y d r o x y l a t i o n . T h e f u n c t i o n o f t h e v a r i o u s c o f a c t o r s was c l a r i f i e d by G r a n t (31) a n d b y Sweat a n d L i p s c o m b (8) who i n d e p e n d e n t l y showed t h a t NADPH was t h e f a c t o r i n v o l v e d i n s t e r o i d 1 1 3 - h y d r o x y l a t i o n a n d t h a t a l l t h e o t h e r compounds w e r e a s s o c i a t e d w i t h t h e i n t r a m i t o c h o n d r i a l p r o d u c t i o n o f t h e r e d u c e d c o e n z y m e . - 4 -I n t h e e n z y m a t i c 1 1 3 - h y d r o x y l a t i o n r e a c t i o n t h e h y d r o x y l g r o u p i n t r o d u c e d d i r e c t l y r e p l a c e s t h e h y d r o g e n a t o m o f t h e same s t e r i c c o n f i g u r a t i o n w i t h o u t a n y i n v e r s i o n o c c u r r i n g ( 3 5 , 3 6 ) . S t u d i e s o n t h e m e c h a n i s m o f h y d r o x y l a t i o n by H a y a n o a n d D o r f m a n (23) i n d i c a t e t h a t t h e A s ( l l ) o r t h e A l l ( l a ) u n s a t u r a t e d a n a l o g u e s o f d e o x y c o r t i c o s t e r o n e a r e n o t i n t e r m e d i a t e s o f 113-h y d r o x y l a t i o n . I n c u b a t i o n s c a r r i e d o u t i n H 2 0 1 8 r e s u l t i n no i n c o r p o r a t i o n o f 0 1 8 i n t o t h e 1 1 3 - h y d r o x y l a t e d p r o d u c t (9). T h u s 1 1 3 - h y d r o x y l a t i o n d o e s n o t i n v o l v e a d e h y d r o g e n a t i o n r e a c t i o n f o l l o w e d by s u b s e q u e n t h y d r a t i o n . H a y a n o et a l . (9) h a v e d e m o n s t r a t e d , u s i n g O i 8 , t h a t t h e o x y g e n o f t h e 113-h y d r o x y l f u n c t i o n i s d e r i v e d t o t a l l y f r o m m o l e c u l a r o x y g e n . T h e 1 1 3 - h y d r o x y l g r o u p i s s t a b l e a n d no e x c h a n g e i s o b s e r v e d when i n c u b a t i o n s a r e c a r r i e d o u t i n t h e p r e s e n c e o f D 20 (23) o r H 2 0 1 8 ( 9 ) . T h e d i r e c t i n v o l v e m e n t o f m o l e c u l a r o x y g e n i n 1 1 3 - h y d r o x y l a t i o n has a l s o b e e n r e p o r t e d b y Sweat et a l . ( 3 7 ) . T h e d e m o n s t r a t i o n o f i n c o r p o r a t i o n o f m o l e c u l a r o x y g e n i n t o t h e s t e r o i d m o l e c u l e e x p l a i n s t h e e a r l i e r o b s e r v a t i o n s t h a t t h i s r e a c t i o n p r o c e e d e d o n l y i n a n o x y g e n a t m o s p h e r e ( 2 2 , 2 4 , 2 5 , 3 4 ) . 1 1 3 - h y d r o x y l a t i o n d o e s n o t p r o c e e d v i a t h e f o r m a t i o n o f a n e:p o x i d e i n t e r m e d i a t e . The f o r m a t i o n o f s m a l l amounts o f t h e 9 3 ,113 - e p - o x i d e f r o m A 9 ^ 1 1 ^ - 1 1 - d e o x y c o r t i s o l h a s b e e n o b s e r v e d (38) b u t t h i s compound i s n o t f u r t h e r m e t a b o l i z e d . T h e s t o i c h i o m e t r i c r e l a t i o n s h i p b e t w e e n s t e r o i d s u b s t r a t e , m o l e c u l a r o x y g e n , a n d NADPH h a s n o t b e e n f i r m l y e s t a b l i s h e d f o r 1 1 3 - h y d r o x y l a t i o n . R e c e n t l y Cammer a n d E s t a b r o o k (39) h a v e - 5 -shown t h a t t h e m o l a r r a t i o o f O2 u p t a k e a n d H B - h y d r o x y l a t i o n i n b e e f a d r e n a l m i t o c h o n d r i a i s u n i t y . C o o p e r et al. (40) h a v e e s t a b l i s h e d t h e s t o i c h i o m e t r y f o r t h e 2 1 - h y d r o x y l a s e o f a d r e n a l m i c r o s o m e s . One m o l e c u l e o f NADPH i s o x i d i z e d f o r e a c h atom o f m o l e c u l a r o x y g e n i n c o r p o r a t e d i n t o t h e C-21 p o s i t i o n o f t h e s t e r o i d s u b s t r a t e . W h i l e t h e c o f a c t o r r e q u i r e m e n t s a n d p r o p e r t i e s o f t h e 118-h y d r o x y l a s e a r e q u i t e w e l l e s t a b l i s h e d , v e r y l i t t l e i s known a b o u t t h i s s y s t e m i n t e r m s o f s u b s t r a t e s p e c i f i c i t y . Sharma et at. (33,41) h a v e shown t h a t t h e 1 1 3 - h y d r o x y l a t i o n o f d e o x y -c o r t i c o s t e r o n e i s i n h i b i t e d c o m p e t i t i v e l y b y 1 1 - d e o x y c o r t i s o l , a n d r o s t e n e d i o n e , t e s t o s t e r o n e , a n d d e h y d r o e p i a n d r o s t e r o n e ; w h e r e a s l l $ - h y d r o x y a n d r o s t e n e d i o n e i s n o t i n h i b i t o r y . I t i s l i k e l y t h a t t h e i n h i b i t o r y e f f e c t o f t h e s e s t e r o i d s i s due t o t h e i r p a r t i c i p a t i o n a s s u b s t r a t e s i n t h e h y d r o x y l a t i o n r e a c -t i o n . S e v e r a l d r u g s a r e a l s o known t o i n h i b i t l l g - h y d r o x y l a -t i o n ( 4 1 - 4 3 ) . M e t o p i r o n e [ 2 - m e t h y l - l , 2 - b i s ( 3 - p y r i d y l ) - 1 -p r o p a n o n e ] i n h i b i t i o n o f H B - h y d r o x y l a t i o n h a s b e e n s t u d i e d e x t e n s i v e l y (42-49) . T h i s compound a p p e a r s t o be r e l a t i v e l y s p e c i f i c f o r m i t o c h o n d r i a l s t e r o i d h y d r o x y l a s e s , e . g . , 11(3-and 1 8 - h y d r o x y l a t i o n . D e t a i l e d s t u d i e s on t h e i n t e r a c t i o n o f s t e r o i d s u b s t r a t e s w i t h t h e 1 1 6 - h y d r o x y l a s e a n d on t h e mode o f a c t i o n o f i n h i b i -t o r s o f t h e h y d r o x y l a t i o n r e a c t i o n may p r o v i d e much i n f o r m a t i o n on t h e o v e r a l l m e c h a n i s m o f t h i s m i x e d f u n c t i o n o x i d a s e . The. mechanisms o f a c t i o n o f two H B - h y d r o x y l a s e i n h i b i t o r s , m e to-p i r o n e a n d d i c u m a r o l , h a v e b e e n e x a m i n e d i n t h i s l a b o r a t o r y - 6 -and t h e r e s u l t s o b t a i n e d a r e p r e s e n t e d i n t h i s t h e s i s . Components o f t h e l l g - H y d r o x y l a s e The f i r s t i n d i c a t i o n o f t h e c o m p l e x n a t u r e o f t h e l l g - h y -d r o x y l a s e was p r o v i d e d by T o m k i n s et al. (50,51) who e x t r a c t e d t h r e e ' h e a t - l a b i l e enzyme f r a c t i o n s f r o m a n a d r e n a l m i t o c h o n d r i a l a c e t o n e p o w d e r , a l l o f w h i c h w e r e r e q u i r e d t o r e c o n s t i t u t e 116-h y d r o x y l a s e a c t i v i t y . The a c t i v i t y c o u l d be s t i m u l a t e d by a d d i t i o n o f a b o i l e d e x t r a c t o f a n a c e t o n e powder o f a d r e n a l o r l i v e r t i s s u e . T h i s h e a t - s t a b l e f a c t o r was d e s t r o y e d o n a s h i n g , was n o t a d s o r b e d o n c h a r c o a l a t a n a c i d pH, and was n e i t h e r t e t r a h y d r o f o l a t e , H 2 0 2 , a s c o r b a t e n o r d i h y d r o x y f u m a r a t e . S w e a t a nd B r y s o n (52) w e r e a b l e t o s e p a r a t e t h e 116-hydroxy-l a s e i n t o two c o m p o n e n t s by ammonium s u l f a t e f r a c t i o n a t i o n . Nakamura a n d a s s o c i a t e s (53,54) d e m o n s t r a t e d t h a t t h e h e a t -s t a b l e f r a c t i o n f i r s t r e p o r t e d b y T o m k i n s was p r o t e i n i n n a t u r e a n d s t i m u l a t e d 116- and 1 8 - h y d r o x y l a t i o n , b u t n o t 2 1 - h y d r o x y -l a t i o n . T h i s h e a t - s t a b l e p r o t e i n h a s b e e n i d e n t i f i e d a s a nonheme i r o n p r o t e i n , a n d has b e e n named a d r e n o d o x i n (55). Omura et al. (56,57) i s o l a t e d a n d p a r t i a l l y p u r i f i e d t h r e e f r a c t i o n s f r o m b e e f a d r e n a l m i t o c h o n d r i a t h a t when com-b i n e d c o u l d r e c o n s t i t u t e 1 1 6 - h y d r o x y l a s e a c t i v i t y . T h e s e t h r e e f r a c t i o n s c o n s i s t e d o f a NADPH f l a v o p r o t e i n r e d u c t a s e , a nonheme i r o n p r o t e i n , a n d a n u n u s u a l h e m o p r o t e i n c a l l e d c y t o c h r o m e P-4 50. They p o s t u l a t e d t h e p a t h w a y f o r h y d o x y l a -t i o n i l l u s t r a t e d i n F i g u r e 1. The i s o l a t i o n a n d i d e n t i f i c a -t i o n o f s i m i l a r c o m p o n e n t s o f t h e r a t and p o r c i n e a d r e n a l * I n d i c a t e s reduced forms of Fp and P i t s o . - 8 -1 1 3 - a n d 1 8 - h y d r o x y T a s e were a l s o r e p o r t e d by Nakamura a nd O t s u k a ( 3 2 ) . The e s t a b l i s h e d c o m p o n e n t s o f t h e 1 1 3 - h y d r o x y l a s e w i l l be d i s c u s s e d i n d i v i d u a l l y . NADPH R e d u c t a s e The f l a v o p r o t e i n t h a t c a t a l y z e s t h e o x i d a t i o n o f NADPH i n a d r e n a l m i t o c h o n d r i a h a s b e e n c a l l e d N A D P H - d i a p o r a s e (57) and a d r e n o d o x i n r e d u c t a s e ( 5 5 , 5 8 , 5 9 ) . T h i s enzyme c a t a l y z e s t h e t r a n s f e r o f e l e c t r o n s f r o m NADPH t o t h e nonheme i r o n c o m p o nent o f t h e 1 1 3 - h y d r o x y l a s e p a t h w a y . I n a d d i t i o n t o t h e n a t u r a l . ' nonheme i r o n p r o t e i n , t h i s f l a v o p r o t e i n may a l s o u t i l i z e f e r r i -c y a n i d e , m e n a d i o n e , a n d c y t o c h r o m e o as e l e c t r o n a c c e p t o r s ( 5 7 , 6 0 ) . NADH i s n o t o x i d i z e d by t h e p u r i f i e d enzyme ( 5 7 ) . T h i s NADPH r e d u c t a s e i s s i m i l a r t o t h e NADPH-cytochrome o r e d u c t a s e o f l i v e r m i c r o s o m e s (61,62) i n t e r m s o f t h e s p e c i f i c compounds i t may u t i l i z e a s e l e c t r o n a c c e p t o r s . Nonheme I r o n P r o t e i n The n a t u r a l e l e c t r o n a c c e p t o r o f t h e a d r e n a l m i t o c h o n d r i a l NADPH r e d u c t a s e i s anonheme i r o n p r o t e i n t h a t h a s b e e n named a d r e n o d o x i n ( 5 5 ) . T h i s p r o t e i n h a s b e e n p u r i f i e d e x t e n s i v e l y by K i m u r a a nd S u z u k i (59) and h a s b e e n shown t o be r e q u i r e d i n 1 1 3 - h y d r o x y l a t i o n ( 5 5 , 5 8 , 6 0 ) . The p u r i f i e d p r o t e i n c o n t a i n s two i r o n atoms and two m o l e s o f a c i d - l a b i l e s u l f i d e p e r m o l e o f p r o t e i n a n d h a s a m o l e c u l a r w e i g h t o f 15,000-20,0 00. Com-b i n a t i o n o f a d r e n o d o x i n a n d NADPH r e d u c t a s e r e c o n s t i t u t e s t h e NADPH-cytochrome P-4 50 r e d u c t a s e a c t i v i t y o f a d r e n a l m i t o c h o n -d r i a ( 5 7 ) ; t h u s a d r e n o d o x i n i s p l a c e d b e t w e e n t h e f l a v o p r o t e i n - 9 -a n d c y t o c h r o m e P - 4 50 i n t h e e l e c t r o n t r a n s p o r t s e q u e n c e p r o p o s e d by Omura et al. ( F i g . 1 ) . A l t h o u g h m i c r o s o m a l h y -d r o x y l a t i o n s a r e b e l i e v e d t o p r o c e e d i n a manner a n a l o g o u s t o t h o s e o f t h e m i t o c h o n d r i a , nonheme i r o n p r o t e i n s i m i l a r t o a d r e n o d o x i n h a s n o t b e e n d e m o n s t r a t e d i n t h i s f r a c t i o n ( 6 3 ) . M i t o c h o n d r i a l a d r e n o d o x i n i s s i m i l a r i n i t s p h y s i c o -c h e m i c a l p r o p e r t i e s t o f e r r e d o x i n ( 6 4 , 6 5 ) a n d t o p h o t o s y n -t h e t i c p y r i d i n e n u c l e o t i d e r e d u c t a s e (66) b u t t h e l a t t e r p r o -t e i n c a n n o t s u b s t i t u t e f o r a d r e n o d o x i n i n l l g - h y d r o x y l a t i o n (58) . C y t o c h r o m e P - 4 5 0 C y t o c h r o m e P - 4 5 0 i s a n u n u s u a l h e m o p r o t e i n t h a t i n t h e r e d u c e d f o r m i s c a p a b l e o f c o m b i n i n g w i t h c a r b o n m o n o x i d e t o f o r m a c o m p l e x e x h i b i t i n g a n a b s o r p t i o n maximum a t 450 my ( 5 6 , 6 7 ) . T h e f i r s t i m p l i c a t i o n f o r a r o l e o f t h i s c y t o c h r o m e i n s t e r o i d h y d r o x y l a t i o n s was p r o v i d e d b y R y a n a n d E n g e l (30) who o b s e r v e d t h a t t h e 2 1 - h y d r o x y l a s e o f b e e f a d r e n a l m i c r o s o m e s was i n h i b i t e d by c a r b o n m o n o x i d e . H o w e v e r t h e y d i d n o t s t u d y t h e n a t u r e o f t h e c a r b o n m o n o x i d e i n h i b i t i o n . E s t a b r o o k et al. (68) d e m o n s t r a t e d d u r i n g s t u d i e s on t h e l i g h t - r e v e r s i b i l i t y o f t h e c a r b o n m o n o x i d e i n h i b i t i o n o f s t e r o i d 2 1 - h y d r o x y l a t i o n t h a t m a x i m a l r e v e r s i b i l i t y o f i n h i b i t i o n was a c h i e v e d a t 450 my, c o r r e s p o n d i n g t o t h e a b s o r p t i o n maximum o f t h e c y t o c h r o m e P - 4 5 0 - c a r b o n m o n o x i d e c o m p l e x . T h e p r e s e n c e o f a s i m i l a r c y t o c h r o m e was o b s e r v e d i n r a t a n d b o v i n e a d r e n a l m i t o c h o n d r i a (67 -71 ) a n d W i l s o n et al. (72) d e m o n s t r a t e d t h a t t h i s hemo-- 10 -p r o t e i n was i n v o l v e d i n s t e r o i d l l g - h y d r o x y l a t i o n . C y t o c h r o m e P - 4 5 0 h a s s i n c e b e e n o b s e r v e d i n a number o f t i s s u e s , i n c l u d i n g l i v e r , k i d n e y , s m a l l i n t e s t i n e , a d r e n a l c o r t e x a n d c o r p u s l u t -eum ( 7 3 - 7 5 ) . C y t o c h r o m e P - 4 5 0 h a s b e e n shown t o p a r t i c i p a t e i n a number o f m i x e d f u n c t i o n o x i d a s e r e a c t i o n s , i n c l u d i n g t h e 1 1 3 - , 1 8 - , a n d 2 1 - h y d r o x y l a t i o n o f s t e r o i d s ( 6 8 , 7 2 , 7 6 , 7 7 ) , t h e c o n v e r s i o n o f c h o l e s t e r o l t o p r e g n e n o l o n e ( 7 8 - 8 0 ) , m i c r o s o m a l d r u g h y d r o x y l a t i o n a n d d e m e t h y l a t i o n ( 8 1 , 8 2 ) , a n d l i p i d m e t a -b o l i s m ( 8 3 ) . I t i s b e l i e v e d t o b e t h e t e r m i n a l o x i d a s e o f m i x e d f u n c t i o n o x i d a s e s y s t e m s . A l l a t t e m p t s t o p u r i f y c y t o c h r o m e P - 4 5 0 h a v e b e e n u n -s u c c e s s f u l b e c a u s e o f t h e e x t r e m e i n s t a b i l i t y o f t h i s hemo-p r o t e i n . T h e r e f o r e t h e p r o p e r t i e s o f t h i s h e m o p r o t e i n h a v e b e e n s t u d i e d m a i n l y b y t h e t e c h n i q u e o f d i f f e r e n c e s p e c t r o -p h o t o m e t r y . T h e r e d u c e d m i n u s o x i d i z e d d i f f e r e n c e s p e c t r u m o f c y t o c h r o m e P - 4 5 0 h a s b e e n d i f f i c u l t t o d e t e r m i n e b e c a u s e o f t h e c o e x i s t a n c e i n m i c r o s o m e s a n d i i i i t r o c h o n d r i a o f o t h e r r e d o x c o m p o n e n t s . N i s h i b a y a s h i a n d S a t o (84) h a v e r e p o r t e d t h a t c y t o c h r o m e P - 4 5 0 o f r a b b i t l i v e r m i c r o s o m e s e x h i b i t s a d i f f e r e n c e s p e c t r u m w i t h two b r o a d a b s o r p t i o n maxima a t 445 a n d 555 my. T h e s e a b s o r p t i o n maxima a r e o f l o w i n t e n s i t y . Cammer a n d E s t a b r o o k (85) o b s e r v e d t h a t t h e d i f f e r e n c e s p e c -t r u m o f c y t o c h r o m e P - 4 5 0 o f b e e f a d r e n a l m i t o c h o n d r i a h a s two a b s o r p t i o n maxima a t 432 a n d 555 my. Upon c o m b i n a t i o n o f t h e r e d u c e d f o r m o f c y t o c h r o m e P - 4 50 w i t h c a r b o n m o n o x i d e a n i n t e n s e a b s o r p t i o n maximum a t 450 my i s o b s e r v e d ( 5 6 , 6 7 , 6 9 ) . T h i s a b s o r p t i o n maximum i s u n i q u e i n t h a t i t o c c u r s a t - 11 -a wa v.e Length h i g h e r t h a n t h a t n o r m a l l y a s c r i b e d t o c a r b o n m o n o x i d e c o m p l e x e s o f h e m O p r o t e i n s ( 5 6 ) . C y t o c h r o m e P - 4 50 may b e i s o l a t e d by s o n i c a t i o n o f m i t o -c h o n d r i a ( 5 6 , 8 6 ) o r by s o l u b i l i z a t i o n o f m i c r o s o m e s w i t h n o n -i o n i c d e t e r g e n t s ( 8 7 ) . C y t o c h r o m e P - 4 5 0 p r e p a r e d by s o n i c a -t i o n o f m i t o c h o n d r i a i s p a r t i c u l a t e i n n a t u r e a n d c a n be s e d i -m e n t e d by u l t r a c e n t r i f u g a t i o n ( 5 6 ) . T h e u n i q u e s p e c t r a l p r o p -e r t i e s o f c y t o c h r o m e P - 4 5 0 a p p e a r t o be m a i n t a i n e d b y t h e s t r u c t u r a l a s s o c i a t i o n o f t h i s h e m o p r o t e i n w i t h m e m b r a n e s . Omura a n d S a t o ( 8 8 , 8 9 ) o b s e r v e d t h a t t r e a t m e n t o f a r a b b i t l i v e r m i c r o s o m a l p r e p a r a t i o n w i t h a 0.1% s o l u t i o n o f s o d i u m d e o x y c h o l a t e o r a h e a t e d s n a k e venom p r e p a r a t i o n r e s u l t e d i n a l o s s o f t h e a b s o r p t i o n maximum a t 450 my i n t h e c a r b o n mono-x i d e d i f f e r e n c e s p e c t r u m o f t h e m i c r o s o m e s w i t h t h e f o r m a t i o n o f a new a b s o r p t i o n maximum a t 420 my. T h i s new maximum was d u e t o a h e m o p r o t e i n t h a t t h e y t e r m e d c y t o c h r o m e P - 4 2 0 . T h e y c o n c l u d e d t h a t s o l u b i l i z a t i o n o f t h e m i c r o s o m e s r e s u l t e d i n t h e c o n v e r s i o n o f c y t o c h r o m e P - 4 5 0 t o P - 4 2 0 . A l a r g e number o f a g e n t s a r e now known t h a t e l i c i t e t h e c o n v e r s i o n o f c y t o -c h r o m e P - 4 5 0 t o P - 4 2 0 . T h e s e i n c l u d e s o d i u m d e o x y c h o l a t e ( 8 8 , 8 9 ) , s n a k e venom ( 8 8 , 8 9 ) , l y s o l e c i t h i n ( 9 0 ) , t r y p s i n ( 8 1 ) , u r e a ( 8 1 ) , b a t h o c u p r o i n e s u l f o n a t e \ (91) , g u a n i d i n e h y d r o c h l o r i d e ( 9 0 ) , n e u t r a l s a l t s ( 9 0 ) , p a r a c h l o r o m e r c u r i b e n z o a t e ( 9 2 ) , a n d o r g a n i c s o l v e n t s ( 8 1 , 9 0 , 9 3 ) . C y t o c h r o m e P - 4 5 0 i s c l o s e l y a s s o c i a t e d w i t h p h o s p h o l i p i d . T h e s p e c i f i c s t r u c t u r a l r e l a t i o n s h i p o f t h e heme t o t h e h y d r o -p h o b i c r e g i o n s o f t h e h e m o p r o t e i n i s m a i n t a i n e d b y t h e c o n -- 12 -f o r m a t i o n o f t h e c y t o c h r o m e P - 4 50 p r o t e i n a n d by t h e p h o s p h o -l i p i d i n t h e e n v i r o n m e n t . I c h i k a w a a n d Yamano (9 3) m a i n t a i n t h a t t h e h y d r o p h o b i c b o n d i n g i n c y t o c h r o m e P - 4 5 0 i s i m p o r t a n t i n t h a t i t b i n d s t h e heme t o t h e a p o p r o t e i n . T h e c o n v e r s i o n o f c y t o c h r o m e P - 4 5 0 t o c y t o c h r o m e P - 4 2 0 r e s u l t s f r o m a d i s t u r -b a n c e o f t h e h y d r o p h o b i c e n v i r o n m e n t a r o u n d t h e heme, e i t h e r t h r o u g h a c o n f o r m a t i o n a l c h a n g e i n t h e h e m o p r o t e i n o r by t h e r e m o v a l o f a h y d r o p h o b i c m o i e t y s u c h as p h o s p h o l i p i d . M a s o n et al. (81) h a v e p r o p o s e d a s t r u c t u r e f o r m i c r o s o m a l c y t o c h r o m e P - 4 5 0 b a s e d o n t h e e f f e c t s o f v a r i o u s a g e n t s o n t h e h e m o p r o t e i n . T h e y s u g g e s t t h a t i n m i x e d - f u n c t i o n o x i d a t i o n , p r o v i s i o n m u s t b e made f o r b o t h o x y g e n a c t i v a t i o n a n d e l e c t r o n t r a n s p o r t a t t h e a c t i v e s i t e , a n d t h a t a c o n f o r m a t i o n a l c h a n g e m u s t o c c u r d u r i n g c a t a l y s i s t o a l l o w t h e s u b s t r a t e , o x y g e n , a n d two r e -d u c i n g e q u i v a l e n t s t o i n t e r a c t . F u r t h e r , t h e s e a u t h o r s p r o -p o s e t h a t c y t o c h r o m e P - 4 50 i s a p h o s p h o l i p i d c o m p l e x o f t h e s u l f i d e o f c y t o c h r o m e P - 4 2 0 ; t h a t i s , t h a t t h e p h o s p h o l i p i d m o i e t y i s l i n k e d t o t h e h e m o p r o t e i n , c y t o c h r o m e P - 4 2 0 , t h r o u g h a s u l f i d e b r i d g e . T h e p h o s p h o l i p i d c o m p o n e n t a f f e c t s t h e p r e s e n t a t i o n o f t h e s u b s t r a t e t o t h e o x i d a s e . S u b s t r a t e , o x y g e n , a n d s u l f i d e i n t e r a c t a t t h e p r o t o h e m e g r o u p , t h e s u l -f i d e s e r v i n g as t h e t e r m i n u s o f a n e l e c t r o n t r a n s p o r t c h a i n t h a t s u p p l i e s t h e r e q u i r e d two r e d u c i n g e q u i v a l e n t s t o t h e m i x e d - f u n c t i o n o x i d a s e . - 13 -C y t o c h r o m e P - 4 20 C y t o c h r o m e P - 4 2 0 , o b t a i n e d b y t r e a t m e n t o f a r a b b i t l i v e r m i c r o s o m a l f r a c t i o n w i t h h e a t e d s n a k e v e n o m , h a s b e e n p u r i -f i e d e x t e n s i v e l y b y Omura a n d S a t o ( 8 9 ) . T h e y o b s e r v e d t h a t t h e r e d u c e d m i n u s o x i d i z e d d i f f e r e n c e s p e c t r u m o f t h e s o l u b l e h e m o p r o t e i n was v e r y s i m i l a r t o t h a t o f c y t o c h r o m e b\ o f b a c t e r i a . U n l i k e t h e b c y t o c h r o m e s h o w e v e r , c y t o c h r o m e P - 4 2 0 , i n i t s r e d u c e d s t a t e , c o m b i n e s w i t h c a r b o n m o n o x i d e , f o r m i n g a c o m p l e x e x h i b i t i n g a n a b s o r p t i o n maximum a t 420 my. A l t h o u g h t h e c y t o c h r o m e P - 4 2 0 p u r i f i e d by Omura a n d S a t o (89) i s s o l u b l e , p a r t i c u l a t e f o r m s a l s o e x i s t ( 9 0 ) . M a s o n et al. (81) h a v e p r e s e n t e d e v i d e n c e f o r t h e e x i s t e n c e o f s e v e r a l s t a t e s o f c y t o c h r o m e P - 4 2 0 . T h e s e f o r m s a l l e x h i b i t t h e same a b s o r p -t i o n s p e c t r a b u t d i f f e r f r o m one a n o t h e r i n t h e i r s p i n s t a t e s as d e t e r m i n e d by e l e c t r o n p a r a m a g n e t i c r e s o n a n c e s p e c t r o s c o p y . I c h i k a w a a n d Yamano (9 4) h a v e o b s e r v e d t h a t d e t e r g e n t a n d s u l f h y d r y l r e a g e n t - p r o d u c e d c y t o c h r o m e P - 4 2 0 c a n b e r e c o n v e r t e d t o c y t o c h r o m e P - 4 5 0 b y t h e a d d i t i o n o f p o l y o l s o r g l u t a t h i o n e . S u c h r e c o n v e r s i o n c o u l d n o t be e f f e c t e d on c y t o c h r o m e P - 4 2 0 p r o d u c e d b y t r e a t m e n t w i t h p h o s p h o l i p a s e , u r e a , o r g u a h i d i n e s a l t s . T h e y h a v e p r o p o s e d a scheme f o r t h e f o r m a t i o n o f v a r i o u s s t a t e s o f c y t o c h r o m e P - 4 2 0 d e p e n d e n t u p o n t h e e x t e n t o f d e g r a d a t i o n o f c y t o c h r o m e P - 4 5 0 ( 9 3 , 9 4 ) . R e m o v a l o f p h o s -p h o l i p i d o r p r o t e i n f r o m c y t o c h r o m e P - 4 5 0 y i e l d s a f o r m o f c y t o c h r o m e P - 4 2 0 w h i c h c a n n o t r e v e r t t o c y t o c h r o m e P - 4 5 0 . T h u s p r o c e d u r e s t h a t t e n d o n l y t o i n t e r f e r e w i t h t h e h y d r o -p h o b i c b o n d i n g i n c y t o c h r o m e P - 4 5 0 p r o d u c e a s t a t e o f c y t o -c h r o m e P - 4 2 0 t h a n c a n be r e c o n v e r t e d t o c y t o c h r o m e P - 4 5 0 . C y t o c h r o m e P - 4 2 0 c a n be e n z y m a t i c a l l y r e d u c e d w i t h NADPH ( 8 8 , 8 9 ) b u t t h e p u r i f i e d c y t o c h r o m e d o e s n o t i n t e r a c t d i r e c t l y w i t h t h e c o e n z y m e . T h e h e m o p r o t e i n c a n a l s o a c c e p t e l e c t r o n s f r o m a d r e n o d o x i n ( 5 9 , 6 0 ) . However i t d o e s n o t f u n c t i o n i n h y d r o x y l a t i o n r e a c t i o n s . T h e h y d r o p h o b i c a l t e r a t i o n a c c o m -p a n y i n g t h e c o n v e r s i o n o f c y t o c h r o m e P - 4 5 0 t o c y t o c h r o m e P - 4 2 0 r e s u l t s i n a c o n c o m i t a n t l o s s o f i t s a b i l i t y t o i n t e r a c t w i t h s u b s t r a t e s f o r h y d r o x y l a t i o n . C y t o c h r o m e P - 4 5 0 a s t h e S u b s t r a t e B i n d i n g S i t e S p e c t r o p h o t o m e t r i c e v i d e n c e i n d i c a t e s t h a t c y t o c h r o m e P - 4 5 0 i s t h e s u b s t r a t e - b i n d i n g s i t e o f m i x e d f u n c t i o n o x i d a s e s ( 9 5 - 9 8 ) . A d d i t i o n o f t h e a p p r o p r i a t e s u b s t r a t e t o m i t o c h o n -d r i a l o r m i c r o s o m a l h y d r o x y l a s e s y s t e m s p r o d u c e s s p e c t r a l c h a n g e s t h a t c a n b e m e a s u r e d b y d i f f e r e n c e s p e c t r o p h o t o m e t r y * . * I m a i a n d S a t o (9 8) h a v e p r e s e n t e d e v i d e n c e t h a t t h e s u b s t r a t e -p r o d u c e d s p e c t r a l c h a n g e s i n v o l v e c y t o c h r o m e P - 4 5 0 a n d t h a t t h e i n t e r a c t i o n o f s u b s t r a t e w i t h t h e h e m o p r o t e i n i s an o b l i g a t o r y s t e p i n t h e m e c h a n i s m o f h y d r o x y l a t i o n . Cammer a n d E s t a b r o o k (85) h a v e n o t e d t h a t d e o x y c o r t i c o s t e r o n e i n d u c e s s p e c t r a l c h a n g e s i n e n z y m a t i c a l l y r e d u c e d c y t o c h r o m e P - 4 5 0 o f a d r e n a l m i t o c h o n d r i a t h a t d i f f e r f r o m t h o s e p r o d u c e d i n o x i d i z e d c y t o c h r o m e P - 4 5 0 . T h e y h a v e o u t l i n e d a s e q u e n c e o f o x i d a t i o n a n d r e d u c t i o n c h a n g e s i n c y t o c h r o m e P - 4 5 0 t h a t o c c u r d u r i n g t h e c o u r s e o f s t e r o i d l l | 3 - h y d r o x y l a t i o n . - 15 -T h e s p e c t r a l c h a n g e s i n c y t o c h r o m e P - 4 5 0 p r o d u c e d by s u b s t r a t e s a r e b e l i e v e d t o r e s u l t f r o m t h e i n t e r a c t i o n o f t h e s u b s t r a t e w i t h t h e p r o t e i n m o i e t y o f t h e h e m o p r o t e i n . T h i s i n t e r a c t i o n p r o d u c e s c o n f o r m a t i o n a l c h a n g e s i n t h e c y t o c h r o m e t h a t c a u s e a n a l t e r a t i o n i n t h e i n t e r a c t i o n o f l i g a n d s w i t h t h e heme . T h i s h y p o t h e s i s i s s u p p o r t e d b y e v i d e n c e o b t a i n e d by e l e c t r o n p a r a m a g n e t i c r e s o n a n c e s p e c -t r o s c o p y t h a t i n d i c a t e s t h a t s u b s t r a t e b i n d i n g m o d i f i e s l i g a n d i n t e r a c t i o n w i t h t h e heme i r o n o f c y t o c h r o m e P - 4 5 0 (99 > 1 0 0 ) . T h e s p e c t r a l c h a n g e s c a n be i n d u c e d b y a number o f s u b s t r a t e s o f t h e m i t o c h o n d r i a l a n d m i c r o s o m a l h y d r o x y l a s e s a s w e l l a s b y some i n h i b i t o r s o f t h e s e h y d r o x y l a s e s a n d b y o r g a n i c s o l v e n t s ( 9 7 , 9 8 ) . T h e t y p e o f s p e c t r a l c h a n g e a p p e a r s t o v a r y w i t h t h e compound e m p l o y e d . S c h e n k m a n et al. (97) h a v e d i v i d e d s u b s t r a t e s a n d i n h i b i t o r s o f t h e m i c r o -s o m a l h y d r o x y l a s e i n t o two c a t a g o r i e s d e p e n d i n g o n t h e t y p e o f s p e c t r a l c h a n g e p r o d u c e d . T h a t d i f f e r e n t s p e c t r a l c h a n g e s a r e e l i c i t e d by d i f f e r e n t s u b s t r a t e s i m p l i e s t h a t c y t o c h r o m e P - 4 5 0 p o s s e s s e s more t h a n one b i n d i n g s i t e f o r s u c h c o m p o u n d s . T h e o b s e r v a t i o n s o n t h e b i n d i n g o f s u b s t r a t e s t o c y t o -c h r o m e P - 4 5 0 suggest .-; t h a t t h e h e m o p r o t e i n may e x i s t i n more t h a n one s t a t e , a n d t h a t c o n v e r s i o n f r o m one s t a t e t o a n o t h e r r e s u l t s f r o m a c o n f o r m a t i o n a l t r a n s i t i o n . S u p p o r t i n g e v i -d e n c e f o r t h e e x i s t e n c e o f more t h a n one f o r m o f c y t o c h r o m e P - 4 5 0 h a s b e e n p r o v i d e d by S a t o a n d a s s o c i a t e s ( 1 0 1 - 1 0 4 ) . C o m b i n a t i o n o f e t h y l i s o c y a n i d e w i t h t h e r e d u c e d f o r m o f m i c r o s o m a l c y t o c h r o m e P - 4 5 0 p r o d u c e s a c o m p l e x e x h i b i t i n g - 16 -a n a b s o r p t i o n s p e c t r u m w i t h maxima a t 430 my a n d 455 my. T h e i n t e n s i t y o f t h e s e a b s o r p t i o n maxima a r e i n f l u e n c e d b y pH i n a n i n t e r d e p e n d e n t m a n n e r . S i m i l a r , b u t much w e a k e r a b s o r p t i o n maxima a r e p r o d u c e d by a n i l i n e , a s u b s t r a t e o f t h e m i c r o s o m a l h y d r o x y l a s e ( 1 0 4 ) . I m a i a n d S a t o (104) h a v e c o n c l u d e d t h a t r e d u c e d c y t o c h r o m e P - 4 5 0 , i n c o m b i n a t i o n w i t h l i p o p h i l i c l i g a n d s s u c h as e t h y l i s o c y a n i d e o r a n i l i n e , e x i s t s i n two i n t e r c o n v e r t i b l e s t a t e s t h a t a r e i n a p H - d e p e n d e n t e q u i l i b r i u m . W h i l e c y t o c h r o m e P - 4 5 0 a p p e a r s t o be t h e s i t e f o r b o t h s u b s t r a t e a n d o x y g e n a c t i v a t i o n i n m i x e d f u n c t i o n o x i d a t i o n , v e r y l i t t l e i s known c o n c e r n i n g t h e i n t e r a c t i o n o f o x y g e n w i t h s u b s t r a t e . On t h e b a s i s o f s t u d i e s w i t h m o d e l c h e m i c a l s y s t e m s , U l l r i c h a n d S t a u d i n g e r (105) s u g g e s t t h a t a f o r m o f t h e o x y g e n a t o m i t s e l f i s t h e h y d r o x y l a t i n g p a r t i c l e i n m i x e d f u n c t i o n o x i d a s e s . O x y g e n i s f i r s t a t t a c h e d t o t h e d i v a l e n t i r o n o f r e d u c e d c y t o c h r o m e P - 4 5 0 (67) w h i c h t h e n i s o x i d i z e d i m m e d i a t e l y . T h e c o m p l e x f o r m e d i s a s t a b i l i z e d f o r m o f O2 ( o r 0 2 H ) w h i c h i s t h e n r e d u c e d by a s e c o n d e l e c t r o n f r o m NADPH. A n i r o n - o x e n e c o m p l e x i s f o r m e d w i t h t h e same o x i d i z -i n g p r o p e r t i e s a s a n o x y g e n a t o m , b u t w i t h one o f t h e two u n p a i r e d e l e c t r o n s a t t a c h e d t o f e r r i c i r o n . W h i l e many o f t h e c o m p o n e n t s o f t h e m i t o c h o n d r i a l a n d m i c r o s o m a l h y d r o x y l a s e s y s t e m s h a v e b e e n i d e n t i f i e d , l i t t l e i s known c o n c e r n i n g t h e s u b s t r a t e s p e c i f i c i t y o f t h e s e s y s t e m s . L i v e r m i c r o s o m a l h y d r o x y l a s e p r e p a r a t i o n s a r e c a p a b l e o f h y d r o x y l a t i n g a w i d e v a r i e t y o f s t r u c t u r a l l y u n r e l a t e d s u b -s t r a t e s . B o t h 1 7 a - a n d 2 1 - h y d r o x y l a s e s a r e p r e s e n t i n t h e - 17 -e n d o p l a s m i c r e t i c u l u m o f a d r e n o c o r t i c a l t i s s u e . I n a d r e n o -c o r t i c a l m i t o c h o n d r i a a t l e a s t t h r e e s t e r o i d r e a c t i o n s a r e known t o i n v o l v e a c y t o c h r o m e P - 4 5 0 , i . e . , 113-/ 1 8 - h y d r o x y l a -t i o n , a n d c h o l e s t e r o l s i d e c h a i n c l e a v a g e . W h e t h e r a s i n g l e c y t o c h r o m e P - 4 5 0 , a n d i t s a s s o c i a t e d e l e c t r o n t r a n s p o r t c h a i n , f u n c t i o n s i n a l l t h r e e r e a c t i o n s i s n o t k n o w n . N a k a m u r a a n d O t s u k a ( 3 2 ) s e p a r a t e d a p r o t e i n c o m p o n e n t o f r a t a d r e n a l m i t o -c h o n d r i a t h a t t h e y r e p o r t e d d e t e r m i n e d t h e c a r b o n a t o m o n t h e s t e r o i d t o be h y d r o x y l a t e d ; t h a t i s , 113- o r 18. F u r t h e r s t u d i e s o n t h i s c o m p o n e n t h a v e n o t b e e n r e p o r t e d , a n d t h e q u e s t i o n w h e t h e r a d d i t i o n a l enzymes o r f a c t o r s e x i s t t h a t c o n f e r s p e c i f i c i t y t o t h e h y d r o x y l a s e r e a c t i o n m u s t a w a i t f u r t h e r i n v e s t i g a t i o n s . T h i s t h e s i s r e p o r t s o n t h e m e c h a n i s m s o f a c t i o n o f two i n h i b i t o r s o f 1 1 3 - h y d r o x y l a t i o n , n a m e l y , d i c u m a r o l a n d M e t o -p i r o n e . . T h e i n t e r a c t i o n s o f t h e i n h i b i t o r s a n d s u b s t r a t e d e o x y c o r t i c o s t e r o n e w i t h t h e 113 - h y d r o x y l a s e p a t h w a y h a v e - b e e n e x a m i n e d . T h e 1 1 3 - h y d r o x y l a t i o n r e a c t i o n s t u d i e d is . o u t l i n e d i n F i g u r e 2 . T h e s t r u c t u r a l f o r m u l a e o f t h e i n h i b i t o r s d i -c u m a r o l a n d M e t o p i r o n e a r e i l l u s t r a t e d i n F i g u r e 3 . E l u c i d a -t i o n o f t h e mechanisnisvof i n h i b i t i o n by t h e s e compounds may l e a d t o a f u r t h e r u n d e r s t a n d i n g o f t h e s p e c i f i c i t y o f t h e 113 - h y -d r o x y l a s e , t h e i n t e r a c t i o n o f s t e r o i d s u b s t r a t e w i t h t h i s s y s t e m , a n d t h e s t r u c t u r a l f e a t u r e s o f b o t h s t e r o i d s u b s t r a t e s a n d i n h i b i t o r s t h a t a l l o w , them t o i n t e r a c t w i t h t h e 113-h y d r o x y l a s e o f a d r e n o c o r t i c a l m i t o c h o n d r i a . - 18 -F i g . 2 . The t r a n s f o r m a t i o n of deoxycorticosterone to c o r t i c o -sterone. - 19 -M A T E R I A L S AND METHODS (a) S o l v e n t s S o l v e n t s (AR g r a d e ) w e r e p u r i f i e d by d i s t i l l a t i o n . A c e t o n e was r e f l u x e d f o r one h o u r w i t h p o t a s s i u m p e r m a n g a n a t e a n d p o t a s -s i u m c a r b o n a t e a n d t h e n d i s t i l l e d t w i c e . E t h a n o l was r e f l u x e d f o r two h o u r s w i t h p o t a s s i u m h y d r o x i d e a n d z i n c a n d t h e n d i s t i l -l e d t w i c e . E t h e r was w a s h e d w i t h a d i l u t e , w e a k l y a c i d i c s o l u -t i o n o f f e r r o u s s u l f a t e (3 x 1 /10 v o l ) , t h e n t o n e u t r a l i t y w i t h d i s t i l l e d w a t e r , a n d d i s t i l l e d f r o m c a l c i u m h y d r i d e u n d e r a n h y -d r o u s c o n d i t i o n s . S p e c t r o s c o p i c g r a d e i s o o c t a n e ( B r i t i s h D r u g H o u s e s , L t d . ) was e m p l o y e d w i t h o u t p u r i f i c a t i o n . (b) C h e m i c a l s T r i s ( h y d r o x y m e t h y l ) a m i n o m e t h a n e ( T r i z m a b a s e , r e a g e n t g r a d e ) was p u r c h a s e d f r o m S i g m a C h e m i c a l C o m p a n y . T r i s b u f f e r was p r e p a r e d b y t i t r a t i n g a s o l u t i o n o f t h i s compound t o t h e d e s i r e d pH w i t h h y d r o c h l o r i c a c i d . NADPH (A g r a d e ) was p u r -c h a s e d f r o m C a l b i o c h e m , I n c . D i c u m a r o l [ 3 , 3 1 - M e t h y l e n e b i s -( 4 - h y d r o x y c o u r a a r i n ) ] was a g i f t o f t h e W i s c o n s i n A g r i c u l t u r a l R e s e a r c h F o u n d a t i o n a n d A b b o t t L a b o r a t o r i e s . A s o l u t i o n ( 0 . 0 1 M) o f d i c u m a r o l was p r e p a r e d by s u s p e n d i n g 25 mg o f t h e c o m -p o u n d i n 2 . 5 m l o f 75% e t h a n o l . A s o l u t i o n o f 1 N p o t a s s i u m h y d r o x i d e was a d d e d d r o p w i s e (4 o r 5 d r o p s ) t o t h e c o n t i n u a l l y warmed s u s p e n s i o n u n t i l a l l t h e d i c u m a r o l d i s s o l v e d . T h e s o l u t i o n was t h e n d i l u t e d t o 7 . 5 m l w i t h d i s t i l l e d w a t e r . M e t o p i r o n e [ 2 - m e t h y l - l , 2 - b i s ( 3 - p y r i d y l ) - 1 - p r o p a n o n e ] was a g i f t o f C i b a Company L i m i t e d . T h i s compound was e m p l o y e d d i s s o l v e d i n a 0 . 1 M s o l u t i o n o f T r i s b u f f e r c o n t a i n i n g 2 . 5 x - 20 -10~ 3 M M g C l 2 (c) S t e r o i d s D e o x y c o r t i c o s t e r o n e , purchased from Sigma C h e m i c a l Company, was p u r i f i e d by c r y s t a l l i z a t i o n s from ace tone-hexane . D e o x y c o r t i c o s t e r o n e - 1 , 2 - 3 H ( T r a c e r l a b ) and d e o x y c o r t i c o s t e r o n e -4 - 1 4 C (New E n g l a n d N u c l e a r C o r p o r a t i o n ) were p u r i f i e d by p a p e r -p a r t i t i o n chromatography i n the s o l v e n t system l i g r o i n - p r o p y -lene g l y c o l (106) and d i l u t e d w i t h r a d i o i n e r t d e o x y c o r t i c o -s t e r o n e to the f i n a l s p e c i f i c a c t i v i t i e s i n d i c a t e d i n the t e x t . R a d i o i n e r t c o r t i c o s t e r o n e , purchased from Mann Research L a b o r a t o r i e s , I n c . , was p u r i f i e d f i r s t by t h i n - l a y e r chroma-tography on s i l i c a g e l G c o n t a i n i n g R a d e l i n GS-115 phosphor i n an e t h y l a c e t a t e s o l v e n t sys tem. Two u l t r a - v i o l e t a b s o r b -i n g zones were o b t a i n e d , one c o r r e s p o n d i n g i n m o b i l i t y to c o r t i c o s t e r o n e , and a second l e s s p o l a r zone p r e s e n t i n a minor amount. The c o r t i c o s t e r o n e zone was s c r a p e d o f f , e l u t e d w i t h m e t h a n o l , and twice c r y s t a l l i z e d from ace tone -hexane . (d) Chromatography Procedures S i l i c a g e l G ( a c c o r d i n g to S t a h l ) c o n t a i n i n g R a d e l i n GS-115 phosphor was employed f o r t h i n - l a y e r chromatography. S i l i c a g e l G was washed w i t h methanol and then d r i e d . R a d e l i n GS-115 phosphor was e x t r a c t e d w i t h methanol i n a s o x h l e t and then d r i e d . To p r e p a r e the chromatography p l a t e s , 50 g of s i l i c a g e l G , 25 mg of R a d e l i n GS-115 phosphor , and 100 ml o f d i s t i l l e d water were mixed t h o r o u g h l y . The s l u r r y was then spread on g l a s s p l a t e s (20 cm-x 20 cm or 10 cm x 20 cm) w i t h - 21 -a D e s a g a a p p a r a t u s . The p l a t e s w e r e t h e n d r i e d i n a n o v e n (^80° C) and s t o r e d i n a d e s i c c a t o r c a b i n e t . P a p e r - p a r t i t i o n 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 w i t h Whatman No. 1 c h r o m a t o g r a m p a p e r t h a t h a d b e e n p r e v i o u s l y w a s h e d w i t h m e t h a n o l t o remove u l t r a - v i o l e t a b s o r b i n g m a t e r i a l . The c h r o m a t o g r a m p a p e r was c u t i n t o s t r i p s , u s u a l l y 2 cm i n w i d t h , f o r u s e i n t h e p a p e r - p a r t i t i o n c h r o m a t o g r a p h y s y s t e m s . (e) I s o l a t i o n o f t h e H B - H y d r o x y l a s e f r o m B e e f A d r e n a l G l a n d s ( i ) P r e p a r a t i o n o f b e e f a d r e n a l m i t o c h o n d r i a : B e e f a d r e n a l g l a n d s o b t a i n e d f r o m t h e s l a u g h t e r h o u s e w e r e f r e e d o f a d h e r i n g f a t a n d c o n n e c t i v e t i s s u e , d i p p e d i n t o a 0.25 M s u c r o s e s o l u t i o n a t 4° C, and s t o r e d a t -20° C u n t i l t h e y w e r e p r o c e s s e d . A l l s t e p s i n t h e p r e p a r a t i o n o f t h e a d r e n a l m i t o c h o n d r i a w e r e c a r r i e d o u t i n t h e c o l d room (4° C ) . The b e e f a d r e n a l g l a n d s , s t i l l p a r t i a l l y f r o z e n , w e r e d i s -r u p t e d i n a 0.25 M s u c r o s e s o l u t i o n (w/v, 1:5) w i t h a W a r i n g b l e n d o r a n d t h e n h o m o g e n i z e d w i t h a P o t t e r - E l v e h j e m t y p e homo-g e n i z e r . The homogenate was c e n t r i f u g e d (1,400 x g f o r 15 m i n u t e s ) and t h e s u p e r n a t a n t w a s . f i l t e r e d t h r o u g h g a u z e a nd c e n t r i f u g e d (10,000 x g f o r 25 m i n u t e s ) t o y i e l d t h e m i t o -c h o n d r i a l p e l l e t . The p e l l e t was w a s h e d t w i c e b y r e s u s p e n s i o n i n t h r e e v o l u m e s o f 0.25 M s u c r o s e s o l u t i o n f o l l o w e d e a c h t i m e by c e n t r i f u g a t i o n a t 12,000 x g f o r 30 m i n u t e s . ( i i ) P r e p a r a t i o n o f t h e m i t o c h o n d r i a l a c e t o n e p o w d e r : The w a s h e d m i t o c h o n d r i a l p e l l e t was s u s p e n d e d i n a m i n i -m a l v o l u m e o f a 0.25 M s u c r o s e s o l u t i o n a n d t h e s u s p e n s i o n was - 22 -poured, w i t h s t i r r i n g , i n t o t e n volumes o f acetone (-2 0° C ) . The m i x t u r e , a f t e r s t a n d i n g a t -20° C f o r f i v e m i n u t e s , was c e n t r i f u g e d and t h e p r e c i p i t a t e was e x t r a c t e d w i t h t h r e e volumes of acetone (-20° C ) . A f t e r t h r e e such e x t r a c t i o n s the p r e c i -p i t a t e was d r i e d in vacuo and s t o r e d over anhydrous c a l c i u m s u l f a t e i n a d e s i c c a t o r a t -10° C. The m i t o c h o n d r i a l a c etone powder c o u l d be s t o r e d f o r a t l e a s t t h r e e months w i t h o u t l o s s o f 1 1 8 - h y d r o x y l a s e a c t i v i t y . A p p r o x i m a t e l y 1 g o f a c e t o n e powder was o b t a i n e d from 100 g o f beef a d r e n a l g l a n d s . The l l $ - h y d r o x y l a s e system was e x t r a c t e d from t h e m i t o -c h o n d r i a l a c e t o n e powder, i m m e d i a t e l y b e f o r e use i n i n c u b a -t i o n s , by t h e method o u t l i n e d by G r a n t ( 3 1 ) . The m i t o c h o n d r i a l a c e tone powder was homogenized i n an i c e - c o l d s o l u t i o n of 0.154 M K C l , t h e homogenate was a l l o w e d t o s t a n d a t 4° C f o r 30 minutes and t h e n c e n t r i f u g e d a t 17,000 x g f o r 30 minutes a t 4° C. I n a l l i n s t a n c e s e x c e p t where o t h e r w i s e i n d i c a t e d , 1 g o f a c e t o n e powder was e x t r a c t e d w i t h 30 ml of a 0.154 M s o l u -t i o n of K C l . I n some p r e l i m i n a r y e x p e r i m e n t s 1 g o f acetone powder was e x t r a c t e d w i t h 10 or 15 ml of a 0.154 M s o l u t i o n of K C l . ( i i i ) P r e p a r a t i o n of t h e H B - h y d r o x y l a s e from s o n i c a t e d a d r e n a l m i t o c h o n d r i a : T h i s H B - h y d r o x y l a s e system was p r e p a r e d a c c o r d i n g t o t h e method o u t l i n e d by Sharma et al. ( 3 3 ) . The washed m i t o c h o n d r i a l p e l l e t was homogenized w i t h f o u r t i m e s i t s w e i g h t o f g l a s s d i s t i l l e d water a t 4° C. The homogenate was th e n s o n i c a t e d f o r 15 minutes a t 4° C i n a B r o n w i l l 20-kc s o n i c o s c i l l a t o r . S o n i c a t i o n was i n t e r r u p t e d b r i e f l y a f t e r 5 and 10 minutes t o - 23 -a l l o w t h e a p p a r a t u s a n d s o n i c a t e t o c o o l . T h e s o n i c a t e was t h e n c e n t r i f u g e d a t 1 0 5 , 0 0 0 x g f o r 60 m i n u t e s . T h e r e s u l t -i n g s u p e r n a t a n t f l u i d was d r i e d b y l y o p h i l i z a t i o n a n d t h e p o w d e r o b t a i n e d was s t o r e d o v e r a n h y d r o u s c a l c i u m s u l f a t e i n a d e s i c c a t o r a t - 1 0 ° C . The y i e l d o f l y o p h i l i z e d p o w d e r f r o m 100 g o f a d r e n a l g l a n d s was 1 .3 g . I n i n c u b a t i o n s w i t h t h i s 1 1 3 - h y d r o x y l a s e p r e p a r a t i o n , t h e d e s i r e d amount o f t h e l y o -p h i l i z e d p o w d e r was w e i g h e d o u t a n d a d d e d d i r e c t l y t o t h e i n c u b a t i o n b u f f e r m i x t u r e . ( f ) P r o t e i n D e t e r m i n a t i o n s T h e amount o f p r o t e i n p r e s e n t i n t h e enzyme p r e p a r a t i o n e x t r a c t e d f r o m t h e m i t o c h o n d r i a l a c e t o n e powder a n d i n t h e enzyme p r e p a r a t i o n f r o m s o n i c a t e d m i t o c h o n d r i a was d e t e r m i n e d b y t h e b i u r e t m e t h o d (10 7) . T h e p r o t e i n c o n c e n t r a t i o n s i n t h e enzyme p r e p a r a t i o n s w e r e e s t i m a t e d b y c o m p a r i s o n t o a s t a n d a r d s o l u t i o n o f b o v i n e s e r u m a l b u m i n . T h e enzyme p r e p a r a t i o n e x t r a c t e d f r o m 1 mg o f m i t o c h o n d r i a l a c e t o n e p o w d e r c o n t a i n s 0 . 2 2 5 mg o f p r o t e i n . One mg o f l y o p h i l i z e d powder f r o m s o n i -c a t e d m i t o c h o n d r i a c o n t a i n s 0 . 2 4 0 mg o f p r o t e i n . (g) G e n e r a l I n c u b a t i o n C o n d i t i o n s I n g e n e r a l , r e a c t i o n s w e r e c a r r i e d o u t i n 25 m l E r l e n -- 3 m e y e r f l a s k s . A 0 . 1 M T r i s s o l u t i o n c o n t a i n i n g 2 . 5 x 10 M MgCl2 was e m p l o y e d a s t h e b u f f e r m e d i u m . T h e pH o f t h e b u f f e r was 7 . 4 . U n l e s s s t a t e d o t h e r w i s e , a l l r e a c t i o n s w e r e c a r r i e d o u t a t 3 7 ° C i n a D u b n o f f m e t a b o l i c s h a k i n g i n c u b a t o r . T h e g a s p h a s e was a i r . - 24 -The o r d e r o f a d d i t i o n o f r e a c t i o n c o m p o n e n t s was g e n e r a l l y as f o l l o w s : s t e r o i d s u b s t r a t e , T r i s b u f f e r , enzyme p r e p a r a t i o n , e x p e r i m e n t a l f a c t o r s , NADPH. D e o x y c o r t i c o s t e r o n e s u b s t r a t e was a d d e d d i s s o l v e d i n p r o p y l e n e g l y c o l . A l t e r n a -t i v e l y t h e s u b s t r a t e was a d d e d t o t h e i n c u b a t i o n f l a s k i n m e t h a n o l , t h e m e t h a n o l was r e m o v e d u n d e r n i t r o g e n , a n d p r o p y -l e n e g l y c o l was t h e n a d d e d t o d i s s o l v e t h e s t e r o i d . O t h e r f a c t o r s o r compounds t o be t e s t e d w e r e d i s s o l v e d o r s u s -p e n d e d i n t h e a p p r o p r i a t e s o l v e n t s a n d a d d e d t o t h e r e a c t i o n f l a s k . P r i o r t o t h e a d d i t i o n o f NADPH,.each r e a c t i o n m i x t u r e was p r e - i n c u b a t e d a t 37° C f o r e i t h e r 5 o r 8 m i n u t e s w i t h s h a k i n g . NADPH was a d d e d i n T r i s b u f f e r medium a t z e r o t i m e . T h r e e g e n e r a l r e a c t i o n s o r p r o p e r t i e s o f t h e 113-h y d r o x y l a s e p r e p a r a t i o n s w e r e e x a m i n e d w i t h r e g a r d t o t h e e f f e c t s o f s t e r o i d s u b s t r a t e s , i n h i b i t o r s ( d i c u m a r o l a n d M e t o p i r o n e ) , a n d o t h e r f a c t o r s o r compounds. T h e s e r e a c t i o n s ( p r o p e r t i e s ) w e r e : (1) NADPH o x i d a t i o n , (2) 1 1 3 - h y d r o x y l a -t i o n , (3) r e d u c t i o n a n d s t a b i l i z a t i o n o f c y t o c h r o m e s P-450 a n d P-420. When e x a m i n i n g t h e e f f e c t s o f t h e v a r i o u s f a c t o r s o r compounds on t h e s e r e a c t i o n s , c o n t r o l i n c u b a t i o n s ( w i t h t h e f a c t o r a b s e n t ) w e r e s i m u l t a n e o u s l y c a r r i e d o u t . The c o n t r o l r e a c t i o n f l a s k r e c e i v e d t h e same amount o f t h e s o l v e n t i n w h i c h t h e t e s t f a c t o r o r compound ( d i s s o l v e d o r s u s p e n d e d ) was a d d e d t o t h e e x p e r i m e n t a l r e a c t i o n f l a s k . (h) S t u d i e s on NADPH O x i d a t i o n NADPH o x i d a t i o n was m e a s u r e d i n a Beckman DU S p e c t r o -p h o t o m e t e r b y t h e d i m i n u t i o n o f a b s o r b a n c e a t 340 mu a g a i n s t - 25 -a b l a n k c o n t a i n i n g a l l i n c u b a t i o n c o m p o n e n t s e x c e p t t h e r e d u c e d c o e n z y m e . A t y p i c a l r e a c t i o n m i x t u r e f o r m e a s u r e m e n t o f NADPH o x i d a t i o n c o n t a i n e d : p r o p y l e n e g l y c o l ( w i t h o r w i t h o u t d e o x y -c o r t i c o s t e r o n e ) , 0 . 0 5 m l ; T r i s - M g C l 2 b u f f e r , 0 . 9 0 m l ; enzyme p r e p a r a t i o n , 2 . 0 m l ; t e s t f a c t o r o r c o m p o u n d , 0 . 0 5 - 0 . 2 m l . T h e s e r e a c t i o n c o m p o n e n t s w e r e p r e - i n c u b a t e d f o r 5 m i n u t e s a n d NADPH (^1 y m o l e ) i n 0 . 1 m l T r i s b u f f e r medium was a d d e d a t z e r o t i m e t o g i v e a f i n a l v o l u m e o f u s u a l l y 3 . 2 m l . The a b s o r b a n c e o f t h e r e a c t i o n m i x t u r e was t h e n m e a s u r e d a t 340 mu a t t i m e i n t e r v a l s , u s u a l l y 5 m i n u t e s , s t a r t i n g a t z e r o t i m e . ( i ) S t u d i e s o n C o r t i c o s t e r o n e F o r m a t i o n ( l l g - H y d r o x y l a t i o n ) C o r t i c o s t e r o n e f o r m a t i o n was a s s a y e d b y t h e i s o l a t i o n a n d s u b s e q u e n t m e a s u r e m e n t o f t h i s compound a f t e r i n c u b a t i o n o f t h e h y d r o x y l a s e s y s t e m w i t h s u b s t r a t e d e o x y c o r t i c o s t e r o n e . When d e o x y c o r t i c o s t e r o n e - 1 , 2 - 3 H o r d e o x y c o r t i c o s t e r o n e - 4 - 1 4 C was e m p l o y e d as t h e s u b s t r a t e , t h e i s o l a t e d c o r t i c o s t e r o n e was a s s a y e d f o r r a d i o a c t i v i t y . I n p r e l i m i n a r y e x p e r i m e n t s n o n -r a d i o a c t i v e d e o x y c o r t i c o s t e r o n e was e m p l o y e d . T h e c o n c e n t r a t i o n s o f r e a c t i o n c o m p o n e n t s , i n c u b a t i o n v o l u m e s , a n d i n c u b a t i o n t i m e s v a r i e d s l i g h t l y w i t h e a c h e x p e r i -m e n t . R e a c t i o n m i x t u r e s w e r e g e n e r a l l y s e t up as f o r t h e s t u d i e s o n NADPH o x i d a t i o n , ; . R e a c t i o n m i x t u r e s w e r e p r e -i n c u b a t e d f o r e i t h e r 5 o r 8 m i n u t e s a n d NADPH was a d d e d a t z e r o t i m e . S t u d i e s w i t h r a d i o a c t i v e d e o x y c o r t i c o s t e r o n e showed t h a t e s s e n t i a l l y no c o n v e r s i o n o f t h i s s u b s t r a t e t o - 26 -c o r t i c o s t e r o n e t o o k p l a c e d u r i n g t h i s p r e - i n c u b a t i o n p e r i o d i n t h e a b s e n c e o f NADPH; t h e amount o f r a d i o a c t i v e c o r t i c o -s t e r o n e i s o l a t e d a f t e r p r e - i n c u b a t i o n was 0.5% o f t h a t i s o l a t e d a f t e r i n c u b a t i o n i n t h e p r e s e n c e o f NADPH. A f t e r t h e d e s i r e d i n c u b a t i o n p e r i o d , r e a c t i o n s w e r e t e r m i n a t e d by t h e a d d i t i o n o f e i t h e r i c e - c o l d c h l o r o f o r m o r e t h y l a c e t a t e (5 o r 15 m l ) . ( i ) K i n e t i c s t u d i e s o n l l g - h y d r o x y l a t i o n : A t y p i c a l i n c u b a t i o n m i x t u r e was s e t up t o c o n t a i n : d e o x y c o r t i c o s t e r o n e - 1 , 2 - 3 H d i s s o l v e d i n 0 . 0 5 m l p r o p y l e n e g l y c o l ; T r i s - M g C l 2 b u f f e r , 0 . 7 0 m l ; enzyme p r e p a r a t i o n , 1 .3 m l ; t e s t f a c t o r o r compound d i s s o l v e d i n 0 . 0 5 m l o f t h e a p p r o -p r i a t e s o l v e n t . T h e m i x t u r e was k e p t i n an i c e - w a t e r b a t h t h r o u g h o u t t h e s e a d d i t i o n s . E a c h c o m p o n e n t ' w a s a d d e d a t i d e n -t i c a l t i m e i n t e r v a l s t o e a c h f l a s k . E a c h r e a c t i o n m i x t u r e was t h e n p r e - i n c u b a t e d w i t h s h a k i n g a t 3 7 ° C f o r 8 m i n u t e s . NADPH was a d d e d i n 0 . 0 5 m l T r i s - M g C l 2 b u f f e r a t z e r o t i m e . A f t e r 90 s e c o n d s o f i n c u b a t i o n , a 1 .0 m l s a m p l e was r e m o v e d f r o m e a c h f l a s k , p i p e t t e d i n t o 2 m l o f i c e - c o l d e t h y l a c e t a t e , a n d m i x e d t h o r o u g h l y w i t h a V o r t e x - G e n i e m i x e r . ( i i ) I s o l a t i o n , p u r i f i c a t i o n , a n d m e a s u r e m e n t o f d e o x y -c o r t i c o s t e r o n e a n d c o r t i c o s t e r o n e : I n some i n s t a n c e s , t h e c h l o r o f o r m - o r e t h y l a c e t a t e -a r r e s t e d r e a c t i o n m i x t u r e s w e r e t r a n s f e r r e d t o s e p a r a t o r y f u n n e l s f o r e x t r a c t i o n . E a c h m i x t u r e was d i l u t e d w i t h w a t e r t o 15 m l a n d t h e n e x t r a c t e d f o u r t i m e s w i t h c h l o r o f o r m o r e t h y l a c e t a t e (15 ml) . T h e c o m b i n e d s o l v e n t f r a c t i o n . : w a s - 27 -e v a p o r a t e d t o d r y n e s s u n d e r r e d u c e d p r e s s u r e i n a warm w a t e r b a t h a n d t h e r e s i d u e r e d i s s o l v e d i n a m i n i m a l amount o f m e t h a n o l p r i o r t o s e p a r a t i o n o f t h e s t e r o i d s . I n t h e k i n e t i c e x p e r i m e n t s , a n d i n t h e t i m e s t u d i e s c a r r i e d o u t when 1 m l s a m p l e s h a d b e e n r e m o v e d a t t i m e i n t e r v a l s f r o m e a c h r e a c t i o n m i x t u r e , e x t r a c t i o n s w e r e c a r r i e d o u t i n 12 m l c e n t r i f u g e t u b e s . T h e r e a c t i o n s a m p l e was e x t r a c t e d f o u r t i m e s w i t h c h l o r o f o r m o r e t h y l a c e t a t e (2 m l ) , a n d t h e s o l v e n t r e m o v e d u n d e r n i t r o g e n . T h e r a d i o a c t i v i t y r e c o v e r e d by b o t h e x t r a c -t i o n p r o c e d u r e s a c c o u n t e d f o r a t l e a s t 95% o f t h e a d d e d s u b -s t r a t e r a d i o a c t i v i t y . I n p r e l i m i n a r y e x p e r i m e n t s o n t h e NADPH r e q u i r e m e n t o f t h e l l g - h y d r o x y l a s e a n d t h e e f f e c t s o f e l e c t r o n t r a n s p o r t i n h i b i t o r s o n 1 1 6 - h y d r o x y l a t i o n , d e o x y c o r t i c o s t e r o n e a n d c o r t i c o s t e r o n e w e r e i s o l a t e d f r o m t h e o r g a n i c s o l v e n t e x -t r a c t s b y t h i n - l a y e r c h r o m a t o g r a p h y o n s i l i c a g e l G . T h 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 o r g a n i c s o l v e n t u s e d i n e x t r a c t i o n was d i s s o l v e d i n a m i n i m a l amount o f m e t h a n o l a n d s p o t t e d o n t h e c h r o m a t o g r a p h y p l a t e . A f t e r d e v e l o p m e n t o f t h e c h r o m a t o g r a m i n e t h y l a c e t a t e , u l t r a - v i o l e t a b s o r b i n g z o n e s c o r r e s p o n d i n g i n m o b i l i t y t o a u t h e n t i c s a m p l e s o f d e o x y c o r t i c o s t e r o n e a n d c o r t i c o s t e r o n e w e r e s c r a p e d o f f a n d e l u t e d w i t h m e t h a n o l . T h e q u a n t i t y o f e l u t e d s t e r o i d was m e a s u r e d s p e c t r o p h o t o m e t r i c a l l y b y i t s a b s o r p t i o n a t o r a b o u t 240 my i n m e t h a n o l s o l u t i o n . When d e o x y c o r t i c o s t e r o n e - 1 , 2 - 3 H o r d e o x y c o r t i c o s t e r o n e -4 - l l + C was e m p l o y e d as t h e s u b s t r a t e , 50 o r 100 y g o f r a d i o i n e r t - 28 -c a r r i e r c o r t i c o s t e r o n e w e r e a d d e d t o t h e o r g a n i c s o l v e n t e x -t r a c t m a t e r i a l a n d t h e m i x t u r e was s u b j e c t e d t o p a p e r - p a r t i t i o n c h r o m a t o g r a p h y i n t h e s o l v e n t s y s t e m t o l u e n e - p r o p y l e n e g l y c o l (108) f o r 18 h o u r s . U n d e r t h e s e c o n d i t i o n s s u b s t r a t e d e o x y -c o r t i c o s t e r o n e was r e c o v e r e d i n t h e s o l v e n t o v e r f l o w . T h e c o r t i c o s t e r o n e z o n e s w e r e l o c a t e d by u l t r a - v i o l e t a b s o r p t i o n , e x c i s e d , a n d e l u t e d w i t h m e t h a n o l . T h e c o r t i c o s t e r o n e e l u a t e s w e r e a s s a y e d f o r r a d i o a c t i v i t y a n d t h e q u a n t i t y o f e l u t e d s t e r o i d was m e a s u r e d s p e c t r o p h o t o m e t r i c a l l y a t 241 my i n m e t h a n o l s o l u t i o n . C h r o m a t o g r a p h y i n t h e s o l v e n t s y s t e m t o l u e n e - p r o p y l e n e g l y c o l u n d e r t h e s e c o n d i t i o n s , r e s o l v e s t h e two s t e r o i d s c o r t i c o s t e r o n e a n d 1 8 - h y d r o x y - d e o x y c o r t i c o s t e r o n e . F o r m a -t i o n o f t h e l a t t e r s t e r o i d h a s b e e n o b s e r v e d i n i n c u b a t i o n s w i t h a d r e n a l m i t o c h o n d r i a ( 2 6 , 4 9 ) . I n some o f t h e p r e s e n t e x p e r i m e n t s , i n c u b a t i o n o f d e o x y c o r t i c o s t e r o n e - 1 , 2 - 3 H r e -s u l t e d i n t h e f o r m a t i o n o f a r a d i o a c t i v e m e t a b o l i t e w i t h a m o b i l i t y s l i g h t l y l e s s t h a n t h a t o f c o r t i c o s t e r o n e i n t h e c h r o m a t o g r a p h y s y s t e m e m p l o y e d . C h e m i c a l i d e n t i f i c a t i o n o f t h i s m e t a b o l i t e was n o t a t t e m p t e d , a l t h o u g h i t s m o b i l i t y was s i m i l a r t o t h a t o f 1 8 - h y d r o x y - d e o x y c o r t i c o s t e r o n e . R a d i o c h e m i c a l p u r i t y o f t h e i s o l a t e d c o r t i c o s t e r o n e was e s t i m a t e d by f o r m a t i o n o f t h e 2 1 - m o n o a c e t a t e . C h e m i c a l f o r m a t i o n o f t h e 2 1 - m o n o a c e t a t e o f c o r t i c o s t e r o n e was a c h i e v e d b y t r e a t i n g t h e s t e r o i d w i t h f o u r d r o p s o f p y r i d i n e -a c e t i c a n h y d r i d e (1 :1 ) f o r two h o u r s a t room t e m p e r a t u r e . T h e r e a c t i o n m i x t u r e was t h e n d i l u t e d w i t h m e t h a n o l a n d - 29 -e v a p o r a t e d t o d r y n e s s u n d e r n i t r o g e n . T h i s s t e p was r e p e a t e d t w i c e . The r e a c t i o n p r o d u c t , a f t e r d r y i n g in vacuo, was c h r o -m a t o g r a p h e d i n t h e s o l v e n t s y s t e m t o l u e n e - p r o p y l e n e g l y c o l . The 2 1 - m o n o a c e t a t e z o n e was e x c i s e d , e l u t e d w i t h m e t h a n o l , and a s s a y e d f o r r a d i o a c t i v i t y . E v i d e n c e f o r r a d i o c h e m i c a l homo-g e n e i t y was p r o v i d e d b y u n a l t e r e d s p e c i f i c a c t i v i t y (cpm/umole) t h r o u g h o u t t h i s p u r i f i c a t i o n p r o c e d u r e . ( i i i ) R a d i o a s s a y m e t h o d : 3H a c t i v i t y o r 1 a c t i v i t y was m e a s u r e d w i t h a N u c l e a r -C h i c a g o m o d e l 725 l i q u i d s c i n t i l l a t i o n c o u n t e r . S a m p l e s o f t h e r a d i o a c t i v e s t e r o i d i n m e t h a n o l s o l u t i o n w e r e p i p e t t e d i n t o l i q u i d s c i n t i l l a t i o n v i a l s a n d t h e m e t h a n o l was re m o v e d in vacuo. The s t e r o i d was t h e n d i s s o l v e d i n 5 m l o f t o l u e n e c o n t a i n i n g PPO ( 0 . 4 % ) , POPOP ( 0 . 0 0 5 % ) , a n d a b s o l u t e e t h a n o l ( 1 % ) . A l l r a d i o a c t i v i t y a s s a y s w e r e p e r f o r m e d i n d u p l i c a t e . The e f f i c i e n c y o f t h e c o u n t i n g m e thod f o r 1 4 C was 86% and f o r 3H was 36-40% . ( j ) S p e c t r o p h o t o m e t r i c A n a l y s i s o f t h e Enzyme P r e p a r a t i o n The t e c h n i q u e o f d i f f e r e n c e s p e c t o s c o p y was e m p l o y e d t o a s s a y t h e a c e t o n e powder enzyme p r e p a r a t i o n f o r c y t o c h r o m e and f l a v i n c o m p o n e n t s . D i f f e r e n c e s p e c t r a w e r e m e a s u r e d i n a U n i c a m SP 800 s p e c t r o p h o t o m e t e r w i t h q u a r t z c u v e t t e s o f 1 cm o p t i c a l p a t h . U n l e s s o t h e r w i s e s t a t e d , d i f f e r e n c e s p e c t r a w e r e r e c o r d e d a t room t e m p e r a t u r e . The s a m p l e t o be a s s a y e d was d i v i d e d e q u a l l y b e t w e e n two c u v e t t e s a n d a b a s e -l i n e was r e c o r d e d . A f t e r a d d i t i o n o f a p p r o p r i a t e a g e n t s t o - 30 -t h e s a m p l e c u v e t t e , t h e s p e c t r u m was t h e n r e c o r d e d . T h e b a s e -l i n e was s u b t r a c t e d f r o m t h e c h a n g e i n l i g h t a b s o r b a n c e p r o -d u c e d o n a d d i t i o n o f t h e a g e n t s t o t h e s a m p l e c u v e t t e a n d t h e r e s u l t a n t d i f f e r e n c e ^ s p e c t r u m was p l o t t e d . I n a t y p i c a l s p e c t r o p h o t o m e t r i c a s s a y o f t h e enzyme p r e -p a r a t i o n , 0 . 1 m l o f p r o p y l e n e g l y c o l , 2 . 0 m l o f T r i s - M g C l 2 b u f f e r m e d i u m , a n d 3 . 9 m l o f t h e enzyme p r e p a r a t i o n w e r e c o m -b i n e d a n d t h e m i x t u r e was d i v i d e d e q u a l l y b e t w e e n two c u v e t t e s . Two t y p e s o f d i f f e r e n c e s p e c t r a w e r e t h e n m e a s u r e d . 1. R e d u c e d - o x i d i z e d d i f f e r e n c e s p e c t r a . A f t e r e s t a b -l i s h i n g a b a s e l i n e f r o m 600 t o 400 my, l i / O y m o l e o f NADPH i n 0 . 0 5 m l o f T r i s - M g C l 2 b u f f e r medium was a d d e d t o t h e s a m p l e c u v e t t e , a n d a n e q u i v a l e n t amount o f T r i s - M g C l 2 b u f f e r medium was a d d e d t o t h e r e f e r e n c e c u v e t t e . The d i f f e r e n c e s p e c t r u m was t h e n r e c o r d e d . I n t h e c h e m i c a l l y p r o d u c e d d i f f e r e n c e s p e c t r a , a few c r y s t a l s o f s o d i u m d i t h i o n i t e w e r e a d d e d i n -s t e a d o f NADPH t o t h e s a m p l e c u v e t t e . 2 . C a r b o n M o n o x i d e d i f f e r e n c e s p e c t r a . P r i o r t o d i v i d i n g t h e r e a c t i o n m i x t u r e b e t w e e n two c u v e t t e s , NADPH o r d i t h i o n i t e was a d d e d t o t h e r e a c t i o n m i x t u r e t o r e d u c e t h e h e m o p r o t e i n s . T h e m i x t u r e was t h e n d i v i d e d e q u a l l y b e t w e e n two c u v e t t t e s a n d a b a s e l i n e r e c o r d e d . C a r b o n m o n o x i d e was t h e n g e n t l y b u b b l e d t h r o u g h t h e s a m p l e c u v e t t e s o l u t i o n f o r one m i n u t e a n d t h e d i f f e r e n c e s p e c t r u m was t h e n r e c o r d e d . (k) Ammonium S u l f a t e F r a c t i o n a t i o n o f t h e Enzyme P r e p a r a t i o n One gram o f a d r e n a l m i t o c h o n d r i a l a c e t o n e p o w d e r was homo-g e n i z e d w i t h 25 m l o f a 0 . 1 5 4 M K C l s o l u t i o n a n d t h e enzyme - 31 -e x t r a c t was o b t a i n e d as p r e v i o u s l y d e s c r i b e d . T h i s enzyme p r e p a r a t i o n was t h e n f r a c t i o n a t e d w i t h ammonium s u l f a t e i n a m a n n e r s i m i l a r t o t h a t d e s c r i b e d b y K i m u r a a n d S u z u k i ( 5 9 ) . T h e p r e p a r a t i o n was m a i n t a i n e d a t 4 ° C t h r o u g h o u t t h e f r a c -t i o n a t i o n p r o c e d u r e . T h e enzyme p r e p a r a t i o n was b r o u g h t t o 20% s a t u r a t i o n by t h e a d d i t i o n o f s o l i d ammonium s u l f a t e . T h e m i x t u r e was a l l o w -ed t o s t a n d f o r t e n m i n u t e s a n d t h e n was c e n t r i f u g e d a t 2 0 , 0 0 0 x g f o r t h i r t y m i n u t e s . T h e s u p e r n a t a n t was d e c a n t e d , b r o u g h t t o 40% s a t u r a t i o n w i t h s o l i d ammonium s u l f a t e , a l l o w e d t o s t a n d f o r t e n m i n u t e s , and t h e n c e n t r i f u g e d a t 2 0 , 0 0 0 x g f o r 30 m i n u t e s . T h e s u p e r n a t a n t was a g a i n d e c a n t e d a n d b r o u g h t t o 80% s a t u r a t i o n w i t h s o l i d ammonium s u l f a t e . A f t e r s t a n d i n g 10 m i n u t e s t h e m i x t u r e was c e n t r i f u g e d a t 2 0 , 0 0 0 x g f o r 30 m i n u t e s a n d t h e s u p e r n a t a n t was d e c a n t e d . T h e p r e c i p i t a t e s o b t a i n e d b e t w e e n 0% a n d 20%, 20% a n d 40%, a n d 40% a n d 80% s a t u r a t i o n w i t h ammonium s u l f a t e w e r e r e -d i s s o l v e d i n 1 . 8 , 3 . 0 , a n d 6 . 0 m l r e s p e c t i v e l y o f a 0 . 0 3 3 M -4 T r i s b u f f e r s o l u t i o n c o n t a i n i n g 8 . 3 x 10 M M g C l 2 a n d 0 . 1 M K C l . The d i t h i o n i t e r e d u c e d m i n u s o x i d i z e d s p e c t r u m a n d t h e d i t h i o n i t e r e d u c e d c a r b o n m o n o x i d e d i f f e r e n c e s p e c t r u m o f e a c h f r a c t i o n were t h e n d e t e r m i n e d . (1) S t u d i e s o n C y t o c h r o m e P - 4 5 0 a n d P - 4 2 0 ( i ) E f f e c t s o f 1 1 3 - h y d r o x y l a s e s u b s t r a t e s , i n h i b i t o r s . . ( D i c u m a r o l o r M e t o p i r o n e ) , a n d i n c u b a t i o n c o n d i t i o n s : C y t o c h r o m e P - 4 5 0 a n d P - 4 2 0 l e v e l s w e r e m e a s u r e d u n d e r - 32 -experimental conditions and i n the presence of agents similar to those employed to measure 113-hydroxylation. It was found that the cytochrome P-450 content varied considerably between d i f f e r e n t enzyme preparations. For this reason control incu-bations were carried out simultaneously with incubations i n the presence of ster o i d and/or of the i n h i b i t o r s dicumarol and Metopirone. Because t h e - quantity of cytochrome P-450 in the enzyme preparation was r e l a t i v e l y low, the t o t a l volume of reaction mixtures employed i n these studies was three times greater than that employed i n the k i n e t i c studies. However the f i n a l concentration of each reaction component was the same. A t y p i c a l reaction mixture for these studies contained: propylene g l y c o l (with or without deoxycorticosterone) 0.15 ml; Tris-MgCl2 buffer mixture, 2.1 ml; enzyme preparation, 3.9 ml; i n h i b i t o r (dicumarol or Metopirone) i n 0.15 ml of the appropriate solvent. The contents of reaction flask were then pre-incubated at 37° C i n a manner analogous to the 113-hydrox y- l a tion studies. In some instances, NADPH i n 0.15 ml of Tris-MgCl2 buffer medium was added and the i n -cubation continued. After the appropriate incubation period s o l i d ammonium sulf a t e was added to give 20% saturation and the mixture was cooled to 4° C. The f r a c t i o n p r e c i p i t a t i n g between 20% and 40% ammonium sulf a t e saturation was then obtained as previously described. The p r e c i p i t a t e was re-dissolved i n 1.5 ml of a 0.033 M solution of T r i s buffer - 4 '• medium containing 8.3 x. 10. M MgCl 2 and 0.1 M KC1. The - 33 -c y t o c h r o m e P - 4 5 0 a n d P - 4 2 0 c o n t e n t o f t h i s m i x t u r e was t h e n d e t e r m i n e d i n t h e f o l l o w i n g m a n n e r . T h e h e m o p r o t e i n s o f e a c h m i x t u r e w e r e r e d u c e d by a d d i t i o n o f a few c r y s t a l s o f s o d i u m d i t h i o n i t e a n d t h e c a r b o n m o n o x i d e d i f f e r e n c e s p e c t r u m was d e t e r m i n e d as p r e v i o u s l y d e s c r i b e d . C y t o c h r o m e P - 4 5 0 was m e a s u r e d b y t h e c h a n g e i n a b s o r b a n c e b e t w e e n 450 my a n d 500 my. C y t o c h r o m e P - 4 2 0 was m e a s u r e d by t h e c h a n g e i n a b s o r b a n c e b e -t w e e n 4 20 my a n d 500 my. ( i i ) S u b s t r a t e a n d i n h i b i t o r - i n d u c e d s p e c t r a l c h a n g e s i n c y t o c h r o m e P - 4 5 0 : T h e enzyme p r e p a r a t i o n f r a c t i o n p r e c i p i t a t i n g b e t w e e n 20% a n d 40% s a t u r a t i o n w i t h ammonium s u l f a t e was e m p l o y e d f o r t h e s t u d i e s o n s p e c t r a l c h a n g e s i n d u c e d i n c y t o c h r o m e P - 4 5 0 b y d e o x y c o r t i c o s t e r o n e , d i c u m a r o l a n d M e t o p i r o n e . B e c a u s e t h e s e s p e c t r a l c h a n g e s w e r e o f l ow i n t e n s i t y a more c o n c e n t r a t e d s o l u t i o n o f t h e 20% t o 40% ammonium s u l f a t e p r e c i p i t a t e was e m p l o y e d . R e a c t i o n m i x t u r e s f o r t h e s e s t u d i e s w e r e s e t up i n t h e f o l l o w i n g m a n n e r . T h e 20% t o 40% ammonium s u l f a t e p r e c i p i t a t e o b t a i n e d f r o m 8 m l o f t h e enzyme p r e p a r a t i o n was d i s s o l v e d i n 1 .6 m l o f a 0 . 0 3 7 5 M -4 T r i s b u f f e r s o l u t i o n c o n t a i n i n g 9 . 4 x 10 M M g C l 2 a n d 0 . 0 9 6 M K C l . T h e m i x t u r e was d i v i d e d e q u a l l y b e t w e e n two c u v e t t e s a n d a b a s e l i n e f r o m 500 my t o 375 my was r e c o r d e d . D e o x y c o r t i c o s t e r o n e d i s s o l v e d i n 0 .0 2 m l o f p r o p y l e n e g l y c o l was a d d e d t o t h e s a m p l e c u v e t t e ; t h e r e f e r e n c e c u v e t t e r e -c e i v e d t h e same q u a n t i t y o f p r o p y l e n e g l y c o l . T h e d i f f e r e n c e - 34 -s p e c t r u m was t h e n r e c o r d e d . The d i f f e r e n c e s p e c t r u m i n d u c e d by t h e a d d i t i o n o f d e o x y c o r t i c o s t e r o n e was f o u n d t o be s t a b l e t h r o u g h o u t t h e p e r i o d o f t i m e d u r i n g w h i c h t h e s e e x p e r i m e n t s w e r e c o n d u c t e d . The a b i l i t y o f t h e l l g - h y d r o x y l a s e i n h i b i t o r s d i c u m a r o l and M e t o p i r o n e t o i n d u c e s p e c t r a l c h a n g e s i n c y t o c h r o m e P-450 was m e a s u r e d i n t h e same manner as t h a t e m p l o y e d f o r d e o x y c o r t i c o s t e r o n e . The e f f e c t s o f d i c u m a r o l a n d M e t o p i r o n e on t h e d e o x y c o r t i c o s t e r o n e - i n d u c e d s p e c t r a l c h a n g e s w e r e m e a s u r e d by two p r o c e d u r e s : 1. A f t e r r e c o r d i n g t h e d e o x y c o r t i c o s t e r o n e - i n d u c e d d i f f e r e n c e s p e c t r u m , d i c u m a r o l o r M e t o p i r o n e i n 0.01 m l o f t h e a p p r o p r i a t e s o l v e n t was a d d e d t o b o t h t h e s a m p l e and r e f e r e n c e c u v e t t e s . The d i f f e r e n c e s p e c t r u m was t h e n a g a i n r e c o r d e d . A c o n t r o l e x p e r i m e n t was c a r r i e d o u t i n w h i c h 0.01 m l o f s o l v e n t a l o n e was a d d e d t o e a c h c u v e t t e . T h i s a d d i t i o n was f o u n d t o h a v e no e f f e c t on t h e m a g n i t u d e o f t h e d e o x y c o r t i c o s t e r o n e - i n d u c e d d i f f e r e n c e s p e c t r u m . 2. P r i o r t o t h e a d d i t i o n o f d e o x y c o r t i c o s t e r o n e , d i -c u m a r o l o r M e t o p i r o n e d i s s o l v e d i n 0.01 m l o f t h e a p p r o p r i a t e s o l v e n t was a d d e d t o b o t h t h e s a m p l e and r e f e r e n c e c u v e t t e s . A b a s e l i n e f r o m 500 my t o 375 my was t h e n r e c o r d e d . Deoxy-c o r t i c o s t e r o n e d i s s o l v e d i n 0.02 m l o f p r o p y l e n e g l y c o l was a d d e d t o t h e s a m p l e c u v e t t e a n d 0.02 m l o f p r o p y l e n e g l y c o l was a d d e d t o t h e r e f e r e n c e c u v e t t e . The d i f f e r e n c e s p e c t r u m was t h e n r e c o r d e d . - 35 -(m) S t u d i e s o n B o v i n e A d r e n a l M i t o c h o n d r i a l L i p i d s ( i ) A c e t o n e - e x t r a c t e d l i p i d f r a c t i o n : The a c e t o n e - e x t r a c t e d l i p i d f r a c t i o n r e f e r s t o t h e m a t e r i a l t h a t i s e x t r a c t e d i n t o a c e t o n e d u r i n g t h e p r e p a r a -t i o n o f t h e a d r e n a l m i t o c h o n d r i a l a c e t o n e p o wder. The a c e t o n e was r e m o v e d f r o m t h e f r a c t i o n u n d e r r e d u c e d p r e s s u r e i n a warm w a t e r b a t h . A l l w a t e r was r e m o v e d f r o m t h e l i p i d f r a c -t i o n by a z e o t r o p i c d i s t i l l a t i o n w i t h a b s o l u t e e t h a n o l . The d r y m a t e r i a l was t h e n g r o u n d i n t o a f i n e powder w i t h a m o r t a r and p e s t l e . F o r s t u d i e s on t h e e f f e c t o f t h i s l i p i d f r a c t i o n on NADPH o x i d a t i o n , l l g - h y d r o x y l a t i o n , o r on c y t o c h r o m e P-450, t h e d r i e d powder was s u s p e n d e d i n a 0.1 M T r i s b u f f e r s o l u -_ 3 t i o n c o n t a i n i n g 2.5 x 10 M M g C l 2 , pH 7.4. The l i p i d c o n -c e n t r a t i o n was u s u a l l y 10 mg/ml. The s u s p e n s i o n was t h e n s o n i c a t e d i n a B r o n w i l l 2 0 - k c s o n i c o s c i l l a t o r f o r 20 m i n u t e s a t 4° C t o d i s p e r s e t h e l i p i d m a t e r i a l . S u c h t r e a t -ment o r i e n t s t h e p h o s p h o l i p i d s t o f o r m m i c e l l e s ( 1 0 9 , 1 1 0 ) . I n c o m p a r a t i v e s t u d i e s w i t h t h e c o m m e r i c a l p h o s p h o l i p i d p r e p a r a t i o n a s o l e c t i n , a d i s p e r s e d s u s p e n s i o n o f t h i s l i p i d was p r e p a r e d i n a s i m i l a r manner. The l i p i d s u s p e n s i o n s w e r e p r e p a r e d i m m e d i a t e l y p r i o r t o u s e i n i n c u b a t i o n m i x -t u r e s . ( i i ) P r e l i m i n a r y f r a c t i o n a t i o n o f t h e a c e t o n e - e x t r a c t e d l i p i d m a t e r i a l : A p r e l i m i n a r y f r a c t i o n a t i o n o f t h e a c e t o n e - e x t r a c t e d l i p i d m a t e r i a l was c a r r i e d o u t a s f o l l o w s . F i v e g o f t h e - 36 -d r i e d l i p i d w e r e e x t r a c t e d w i t h 50 m l o f p e t r o l e u m e t h e r ( b . p . 4 0 ° - 6 0 ° C) b y c o n t i n u o u s s h a k i n g o f t h e l i p i d - s o l v e n t m i x t u r e f o r two h o u r s w i t h a B u r e l l w r i s t a g i t a t o r . T h e s u s p e n s i o n was t h e n c e n t r i f u g e d , t h e s u p e r n a t a n t d e c a n t e d , a n d t h e p r e c i p i t a t e was r e - e x t r a c t e d w i t h a n a d d i t i o n a l 50 m l o f p e t r o l e u m e t h e r . The c o m b i n e d p e t r o l e u m e t h e r e x t r a c t was" c o n c e n t r a t e d t o a v o l u m e o f 20 m l u n d e r r e d u c e d p r e s s u r e a n d t h e c o n c e n t r a t e was p o u r e d , w i t h s t i r r i n g , i n t o 200 m l o f a c e t o n e p r e v i o u s l y c h i l l e d t o - 2 0 ° C . T h e m i x t u r e was a l l o w e d t o s t a n d o v e r n i g h t a t - 2 0 ° C . T h e p r e c i p i t a t e t h a t f o r m e d was r e m o v e d by c e n t r i f u g a t i o n a n d t h e a c e t o n e was e v a p o r a t e d f r o m t h e s u p e r n a t a n t u n d e r r e d u c e d p r e s s u r e . A p o r t i o n o f e a c h f r a c t i o n o b t a i n e d i n t h i s s e p a r a t i o n p r o c e d u r e was e x a m i n e d f o r i t s e f f e c t o n NADPH o x i d a t i o n a n d 1 1 3 - h y d r o x y -l a t i o n . E a c h s a m p l e was d r i e d in vacuo, a n d an e q u i v a l e n t amount o f t h e d r i e d m a t e r i a l f r o m e a c h f r a c t i o n was d i s p e r s e d i n T r i s-MgCl2 b u f f e r medium by u l t r a s o n i c t r e a t m e n t . ( i i i ) E x t r a c t i o n o f t h e t o t a l l i p i d s o f a d r e n a l m i t o -c h o n d r i a : The p r o c e d u r e e m p l o y e d f o r t h e e x t r a c t i o n a n d i s o l a t i o n o f t h e a d r e n a l m i t o c h o n d r i a l l i p i d s was s i m i l a r t o t h e m e t h o d d e s c r i b e d by C r a n e et al. ( I l l ) f o r t h e i s o l a t i o n o f c o e n z y m e Q f r o m b e e f h e a r t m i t o c h o n d r i a . S e v e n t y g w e t w e i g h t o f m i t o c h o n d r i a , o b t a i n e d f r o m 1000 g o f w h o l e b e e f a d r e n a l s , w e r e s u s p e n d e d i n a 0 . 2 5 M s o l u t i o n o f s u c r o s e t o g i v e a t o t a l v o l u m e o f 150 m l . T h e s u b s e q u e n t - 37 -e x t r a c t i o n a n d f r a c t i o n a t i o n o f l i p i d s w e r e c a r r i e d o u t a t room t e m p e r a t u r e . The m i t o c h o n d r i a l s u s p e n s i o n was p o u r e d i n t o 1,500 m l o f an e t h a n o l - e t h e r m i x t u r e ( 3 : 1 v/v) a n d t h e m i x t u r e was s t i r r e d c o n t i n u o u s l y f o r one h o u r . S o l i d s w e r e f i l t e r e d o f f on a B u c h n e r f u n n e l a n d t w i c e r e - e x t r a c t e d w i t h e t h a n o l - e t h e r b y t h e same p r o c e d u r e . The c o m b i n e d e t h a n o l -e t h e r e x t r a c t was e v a p o r a t e d u n d e r r e d u c e d p r e s s u r e i n a warm w a t e r b a t h u n t i l t h e t o t a l v o l u m e was 250 m l . T h i s a q u e o u s e t h a n o l s u s p e n s i o n (250 ml) was t h e n e x t r a c t e d w i t h p e t r o -l e u m e t h e r , b . p. 40° - 60° C, (3 x 150 ml) and t h e c o m b i n e d p e t r o l e u m e t h e r e x t r a c t was r e d u c e d t o a v o l u m e o f 15 m l u n d e r r e d u c e d p r e s s u r e . T h i s e x t r a c t was t h e n p o u r e d , w i t h s t i r r i n g , i n t o 300 m l o f a c e t o n e and t h e m i x t u r e was s t o r e d a t -20° C o v e r n i g h t . The a c e t o n e s u s p e n s i o n was t h e n f i l -t e r e d i n t h e c o l d t o remove p r e c i p i t a t e d p h o s p h o l i p i d . The f i l t r a t e was e v a p o r a t e d u n d e r r e d u c e d p r e s s u r e a n d t h e r e s i d u e was r e d i s s o l v e d i n 20 m l o f a b s o l u t e e t h a n o l w i t h w a r m i n g . The e t h a n o l s o l u t i o n was s t o r e d o v e r n i g h t a t -20° C a n d t h e w h i t e c r y s t a l l i n e m a t e r i a l o b t a i n e d ( c h o l e s t e r o l ) was t h e n r e m o v e d b y c e n t r i f u g a t i o n . E a c h f r a c t i o n o b t a i n e d b y t h i s p r o c e d u r e was e x a m i n e d f o r i t s e f f e c t o n NADPH o x i d a t i o n b y t h e enzyme p r e p a r a t i o n . An e q u i v a l e n t p e r c e n t a g e o f e a c h f r a c t i o n was r e m o v e d , e v a p o r a t e d t o d r y n e s s and t h e m a t e r i a l r e d i s s o l v e d i n e t h a n o l . A p o r t i o n o f e a c h e t h a n o l f r a c t i o n was t h e n a d d e d t o r e a c t i o n m i x t u r e s e m p l o y e d i n t h e m e a s u r e m e n t o f NADPH o x i d a t i o n . - 38 -( i v ) S i l i c a g e l c o l u m n c h r o m a t o g r a p h y o f t h e l i p i d f r a c -t i o n s o l u b l e i n e t h a n o l a t - 2 0 ° C : A s i l i c a g e l c o l u m n was p r e p a r e d by m i x i n g a p p r o x i m a t e l y 13 g o f s i l i c a g e l ( D a v i s o n g r a d e 9 2 3 , mesh s i z e 100-200) w i t h h e x a n e f o l l o w e d by p o u r i n g t h e s l u r r y i n t o a g l a s s c o l u m n h a l f - f i l l e d w i t h h e x a n e . T h e s i l i c a g e l was a l l o w e d t o s e t t l e a n d t h e e x c e s s h e x a n e was d r a i n e d o f f . T h e f i n a l d i m e n s i o n s o f t h e s i l i c a g e l c o l u m n w e r e 1 cm x 19 c m . T h e - 2 0 ° C e t h a n o l - s o l u b l e f r a c t i o n o b t a i n e d by f r a c t i o n a t i o n o f t h e t o t a l m i t o c h o n d r i a l l i p i d s f r o m 70 g w e t w e i g h t o f a d r e n a l m i t o c h o n d r i a was e v a p o r a t e d t o d r y n e s s u n d e r r e d u c e d p r e s s u r e , r e d i s s o l v e d i n 15 m l o f h e x a n e , a n d a d d e d t o t h e c o l u m n . T h e c o l u m n was t h e n d e v e l o p e d w i t h t h e f o l l o w i n g s o l v e n t s o r s o l v e n t c o m b i n a t i o n s : 250 m l o f h e x a n e , 50 m l o f h e x a n e - c h l o r o f o r m ( 1 9 : 1 ) , 50 m l o f h e x a n e - c h l o r o f o r m ( 9 : 1 ) , 50 m l o f h e x a n e - c h l o r o f o r m ( 4 : 1 ) , 50 m l o f h e x a n e - c h l o r o f o r m ( 1 . 5 : 1 ) , 150 m l o f c h l o r o f o r m , a n d 100 m l o f m e t h a n o l . F i f t y m l e l u a t e f r a c t i o n s w e r e c o l l e c t e d . T h e s o l v e n t was r e m o v e d f r o m e a c h f r a c t i o n u n d e r r e d u c e d p r e s s u r e a n d e a c h r e s i d u e was r e d i s s o l v e d i n 5 . 0 m l o f 95% e t h a n o l . E q u i v a l e n t p o r -t i o n s o f e a c h f r a c t i o n w e r e t h e n a s s a y e d f o r t h e i r e f f e c t on NADPH o x i d a t i o n . A p o r t i o n o f e a c h c o l u m n e l u a t e was a p p l i e d t o a t h i n -l a y e r c h r o m a t o g r a p h y p l a t e o f s i l i c a g e l G . T h e c h r o m a t o -g r a p h y p l a t e was d e v e l o p e d w i t h b e n z e n e . A s a m p l e o f c o m -m e r c i a l c o e n z y m e Q i o (Mann R e s e a r c h L a b o r a t o r i e s ) was r u n as a s t a n d a r d . T h e d e v e l o p e d c h r o m a t o g r a m was e x a m i n e d f o r - 39 -u l t r a - v i o l e t a b s o r b i n g z o n e s a n d was t h e n s p r a y e d w i t h a s o l u -t i o n o f l e u c o m e t h y l e n e b l u e . Z o n e s c a p a b l e o f u n d e r g o i n g r e -d u c t i o n a p p e a r e d as b l u e s p o t s . R e d u c e d ( l e u c o ) m e t h y l e n e b l u e was p r e p a r e d by t h e m e t h o d o f C r a n e a n d D i l l e y ( 1 1 2 ) . 50 mg o f m e t h y l e n e b l u e w e r e d i s s o l v e d i n 50 m l o f 95% e t h a n o l . 0 . 5 g o f z i n c d u s t a n d 0 . 5 m l o f g l a c i a l a c e t i c a c i d w e r e a d d e d , t h e m i x t u r e s w i r l e d u n t i l c o l o u r l e s s a n d t h e n f i l t e r e d t h r o u g h g l a s s w o o l . T h i s r e a g e n t was t h e n s p r a y e d o n t h e t h i n - l a y e r c h r o m a t o g r a p h y p l a t e . (v) S a p o n i f i c a t i o n o f t h e a d r e n a l m i t o c h o n d r i a l a c e t o n e p o w d e r : T h e p o s s i b l e p r e s e n c e o f b o u n d l i p o q u i n o n e s i n t h e m i t o -c h o n d r i a l a c e t o n e p o w d e r was e x a m i n e d by s a p o n i f i c a t i o n o f t h e p r e p a r a t i o n a c c o r d i n g t o t h e m e t h o d o f G a l e et al. ( 1 1 3 ) . F o u r g o f a d r e n a l m i t o c h o n d r i a l a c e t o n e p o w d e r , 2 . 0 g o f p y r o g a l l o l , 8 g o f s o d i u m h y d r o x i d e , 40 m l o f m e t h a n o l a n d 30 m l o f w a t e r w e r e a d d e d t o a 250 m l r o u n d b o t t o m f l a s k a n d t h e m i x t u r e was r e f l u x e d f o r one h o u r . A f t e r c o o l i n g s l i g h t l y , t h e s t i l l warm r e f l u x m i x t u r e was e x t r a c t e d w i t h h e x a n e (3 x 150 ml) . T h e c o m b i n e d h e x a n e e x t r a c t was c o n c e n t r a t e d t o 50 m l u n d e r r e d u c e d p r e s s u r e . T h e c o n c e n t r a t e was t h e n w a s h e d w i t h w a t e r (3 x 50 m l ) , d r i e d o v e r a n h y d r o u s s o d i u m s u l f a t e , f i l t e r e d , a n d e v a p o r a t e d t o d r y n e s s u n d e r r e d u c e d p r e s s u r e . T h i s m a t e r i a l was t h e n a s s a y e d f o r l i p o q u i n o n e s a c c o r d i n g t o t h e p r o c e d u r e o u t l i n e d b y B r a g g a n d P o l g l a s e ( 1 1 4 ) . T h e h e x a n e - e x t r a c t e d m a t e r i a l was d i s s o l v e d i n 5 m l o f i s o o c t a n e a n d a p p l i e d t o a M a g n e s o l - C e l i t e (5:1) c o l u m n , t h e d i m e n s i o n s o f w h i c h w e r e 1 cm x 3 cm. T h e c o l u m n was - 40 -e l u t e d w i t h the f o l l o w i n g solvents or s o l v e n t combinations: 20 ml of isooctane, 20 ml of e t h e r - i s o o c t a n e (1:99), 2 x 20 ml of ethanol-isooctane (1:99), and 20 ml of acetone. Each el u a t e was evaporated to dryness, the m a t e r i a l r e d i s -solved i n 2 ml of isooctane and then assayed spectrophoto-m e t r i c a l l y from 340 to 230 my f o r the presence of lipoquinones. - 41 -PART I GENERAL PROPERTIES OF THE 113-HYDROXYLASE EXTRACTED FROM A BOVINE ADRENAL MITOCHONDRIAL ACETONE POWDER The enzyme sytem which c a t a l y z e s the i n t r o d u c t i o n of a hydroxyl group i n t o the 113 p o s i t i o n of the s t e r o i d nucleus i s l o c a t e d i n the m i t o c h o n d r i a l f r a c t i o n of a d r e n o c o r t i c a l t i s s u e . The 1 1 3~hydroxylase has been i s o l a t e d by a v a r i e t y of methods (31-33) from a d r e n a l t i s s u e . The enzyme employed i n the c u r r e n t s t u d i e s was e x t r a c t e d from a bovine a d r e n a l m i t o c h o n d r i a l acetone powder with a 0.154 M s o l u t i o n of KC1. The g e n e r a l p r o p e r t i e s of the system i s o l a t e d i n t h i s .manner were s t u d i e d . RESULTS (a) NADPH Requirement of the 113-Hydroxylase The s t e r o i d 113-hydroxylase i s a member of the g e n e r a l group of enzymes termed mixed f u n c t i o n oxidases t h a t c a t a -l y z e the i n t r o d u c t i o n of an atom of molecular oxygen i n t o the s u b s t r a t e molecule concomitant w i t h the o x i d a t i o n of NADPH. The dependence of 1 1 3-hydroxylation on NADPH i n the enzyme p r e p a r a t i o n employed i n our s t u d i e s i s shown i n Table I. No c o n v e r s i o n o f d e o x y c o r t i c o s t e r o n e to c o r t i -c o sterone occurs i n the absence of added NADPH. Indeed the data i n d i c a t e t h a t e x t e n s i v e c o n v e r s i o n takes p l a c e o n l y a t high molar r a t i o s of NADPH to d e o x y c o r t i c o s t e r o n e . For example, 10 umoles of NADPH are r e q u i r e d f o r the formation of 0.37 ymole of c o r t i c o s t e r o n e . That no c o r t i c o s t e r o n e can be - 42 -TABLE I E f f e c t o f i n c r e a s i n g q u a n t i t i e s o f NADPH on t h e '11(3-hydroxy-l a t i o n o f d e o x y c o r t i c o s t e r o n e NADPH a d d e d ( u m o l e s ) D e o x y c o r t i c o s t e r o n e • - r e c o v e r e d (umole) C o r t i c o s t e r o n e s y n t h e s i z e d Cumole) P e r c e n t c o n v e r s i o n 0 0. 430 0 0 5 0. 214 .0. 213 44 7.5 0.078 0.325 67 10 0.014 0.365 75 E a c h r e a c t i o n f l a s k c o n t a i n e d : 0.484 umole o f d e o x y c o r -t i c o s t e r o n e d i s s o l v e d i n 0.10 m l o f p r o p y l e n e g l y c o l ; 1.50 ml o f T r i s - M g C l 2 b u f f e r ; 3 m l o f enzyme p r e p a r a t i o n Cl g o f m i t o -c h o n d r i a l a c e t o n e powder e x t r a c t e d w i t h 10 m l o f a 0.154 M K C l s o l u t i o n ) ; NADPH d i s s o l v e d i n 0.5 m l o f T r i s - M g C l 2 b u f f e r . . The t o t a l v o l u m e was 5.1 m l . A f t e r 9 0 m i n u t e s o f i n c u b a t i o n , r e a c -t i o n s w e r e t e r m i n a t e d b y t h e a d d i t i o n o f e t h y l a c e t a t e Q.5 ml) t o e a c h f l a s k . - 43 -d e t e c t e d i n t h e a b s e n c e o f a d d e d NADPH e x c l u d e s t h e p r e s e n c e o f e n d o g e n o u s c o r t i c o s t e r o n e i n t h e l l g - h y d r o x y l a s e p r e p a r a t i o n . P e r o n a n d a s s o c i a t e s (115,116) f o u n d i n s t u d i e s w i t h i n -t a c t m i t o c h o n d r i a t h a t NADPH s u p p o r t e d o n l y a r e l a t i v e l y l o w r a t e o f l l g - h y d r o x y l a t i o n when c o m p a r e d w i t h t h a t b y K r e b s c y c l e i n t e r m e d i a t e s . S i m i l a r l y H a r d i n g et al. (117) f o u n d t h a t s u c c i n a t e and m a l a t e s u p p o r t e d a much h i g h e r r a t e o f 1 1 3 - h y d r o x y l a t i o n t h a n d i d NADPH. B o t h g r o u p s o f w o r k e r s s u g g e s t e d t h a t t h e f u n c t i o n o f t h e t r i c a r b o x y l i c a c i d c y c l e i n t e r m e d i a t e s was t o g e n e r a t e i n t r a m i t o c h o n d r i a l NADPH r e -q u i r e d f o r l l | 3 - h y d r o x y l a t i o n . They a t t r i b u t e d t h e i n a b i l i t y o f e x o g e n o u s NADPH t o s u p p o r t 1 1 3 - h y d r o x y l a t i o n t o t h e i m -p e r m e a b i l i t y o f m i t o c h o n d r i a t o r e d u c e d p y r i d i n e n u c l e o t i d e s ( 1 1 5 - 1 1 9 ) . I n d e e d P e r o n et al. (116 , 1 2 0 ) showed t h a t NADPH -s u p p o r t e d 1 1 3 - h y d r o x y l a t i o n c o u l d be s t i m u l a t e d b y t h e a d d i -t i o n o f c a l c i u m a n d c o n c l u d e d t h a t m i t o c h o n d r i a l s w e l l i n g r e s u l t i n g f r o m t h e a d d i t i o n o f c a l c i u m a l l o w e d e x o g e n o u s NADPH t o e n t e r t h e m i t o c h o n d r i a a n d t h u s s u p p o r t 1 1 3 - h y d r o x y -l a t i o n . The enzyme p r e p a r a t i o n u s e d i n o u r s t u d i e s was a n e x t r a c t o f a m i t o c h o n d r i a l a c e t o n e p owder. T h e r e f o r e t h e i m p e r m e a b i l i t y o f NADPH w o u l d n o t be a l i m i t i n g f a c t o r a n d c o u l d n o t a c c o u n t f o r t h e h i g h c o n c e n t r a t i o n s o f NADPH r e q u i r e d b y t h i s p r e p a r a t i o n f o r 1 1 3 - h y d r o x y l a t i o n . The r e q u i r e m e n t o f t h e enzyme p r e p a r a t i o n f o r h i g h c o n -c e n t r a t i o n s of-NADPH s u g g e s t e d t h a t t h e c o f a c t o r was b e i n g u t i l i z e d i n r e a c t i o n s o t h e r t h a n 1 1 3 - h y d r o x y l a t i o n . O x i d a t i o n o f NADPH by t h e enzyme p r e p a r a t i o n i n b o t h t h e a b s e n c e a n d - 44 -p r e s e n c e o f s t e r o i d s u b s t r a t e was t h e r e f o r e e x a m i n e d . The r e s u l t s a r e shown i n F i g . 4. I t i s a p p a r e n t t h a t t h e enzyme p r e p a r a t i o n c a t a l y z e s t h e r a p i d d i s a p p e a r a n c e o f t h e r e d u c e d coenzyme i n t h e a b s e n c e o f s t e r o i d s u b s t r a t e . A d d i t i o n o f d e o x y c o r t i c o s t e r o n e t o t h e r e a c t i o n m i x t u r e r e s u l t s i n an i n -c r e a s e d r a t e o f NADPH o x i d a t i o n . The " b a c k g r o u n d " o x i d a t i o n o f NADPH i n t h e a b s e n c e o f s t e r o i d i s r e s p o n s i b l e f o r 83% o f t h e t o t a l NADPH o x i d i z e d i n t h e p r e s e n c e o f s t e r o i d d u r i n g a 90 m i n u t e i n c u b a t i o n p e r i o d a n d l i k e l y a c c o u n t s f o r t h e h i g h m o l a r r a t i o s o f NADPH t o s t e r o i d t h a t a r e r e q u i r e d f o r s i g n i f i c a n t h y d r o x y l a t i o n . 1 1 3 - h y d r o x y l a t i o n o f d e o x y c o r t i c o s t e r o n e was m e a s u r e d t h r o u g h o u t t h e p e r i o d o f NADPH o x i d a t i o n shown i n F i g u r e 4. The p e r c e n t c o n v e r s i o n o f d e o x y c o r t i c o s t e r o n e t o c o r t i c o s t e r o n e a s a f u n c t i o n o f t i m e i s shown i n T a b l e I I . H y d r o x y l a t i o n p r o c e e d s m o s t r a p i d l y d u r i n g t h e f i r s t 45 m i n u t e s o f i n c u b a -t i o n . A maximum o f 70% c o n v e r s i o n o f d e o x y c o r t i c o s t e r o n e t o c o r t i c o s t e r o n e i s a c h i e v e d a f t e r 60 m i n u t e s o f i n c u b a t i o n a n d no s i g n i f i c a n t i n c r e a s e i s o b s e r v e d a f t e r t h i s t i m e e v e n t h o u g h NADPH o x i d a t i o n i s s t i l l p r o c e e d i n g ( F i g . 4 ) . The d e c r e a s e d r a t e o f c o r t i c o s t e r o n e f o r m a t i o n i n d i c a t e s t h a t p o s s i b l y some o t h e r f a c t o r i n v o l v e d i n 1 1 3 - h y d r o x y l a t i o n i s b e c o m i n g r a t e l i m i t i n g , o r t h a t an enzyme i n t h e 1 1 3 -h y d r o x y l a s e s y s t e m i s u n s t a b l e t o p r o l o n g e d i n c u b a t i o n . The d e c r e a s e d r a t e o f 1 1 3 - h y d r o x y l a t i o n may a l s o be t h e r e s u l t o f i n h i b i t i o n o f t h e r e a c t i o n by t h e p r o d u c t c o r t i c o s t e r o n e . C o m p a r i s o n o f t h e NADPH o x i d a t i o n d i r e c t l y a t t r i b u t a b l e - 45 -7 INCUBATION T IME , min F i g . 4. O x i d a t i o n of NADPH i n the presence and absence of deoxy c o r t i c o s t e r o n e . Incubations were c a r r i e d out as described i n Table I I . P r i o r to the a d d i t i o n of e t h y l a c e t a t e , 0.2 ml of each reac-t i o n mixture was removed, d i l u t e d to 4 ml with water, and the absor p t i o n at 340 my was measured. NADPH o x i d a t i o n i n the absence of deoxycorticosterone was measured a f t e r 0, 15, 30, 45, 60, and 90 minutes of i n c u b a t i o n by removing 0.2 ml por-t i o n s of a r e a c t i o n mixture c o n t a i n i n g 0.1 ml propylene g l y -c o l i n s t e a d of deox y c o r t i c o s t e r o n e . C o n t r o l (©); plus deoxycorticosterone (o). - 46 -TABLE I I l l g - h y d r o x y l a t i o n o f d e o x y c o r t i c o s t e r o n e a s a f u n c t i o n o f t i m e I n c u b a t i o n t i m e ( m i n u t e s ) D e o x y c o r t i c o s t e r o n e r e c o v e r e d (ymole) C o r t i c o s t e r o n e s y n t h e s i z e d ( y mole) P e r c e n t c o n v e r s i o n 15 0.372 0.246 30.8 30 0.251 0.375 47.1 45 0.106 0.517 64.8 60 0.039 0.563 70.5 75 0.030 0.568 71.3 90 0.036 0.573 71.8 E a c h r e a c t i o n f l a s k c o n t a i n e d : 0.8 00 y m o l e o f d e o x y c o r t i -c o s t e r o n e d i s s o l v e d i n 0.1 m l o f p r o p y l e n e g l y c o l ; 1.50 m l o f T r i s - M g C l 2 b u f f e r ; 3 m l o f enzyme p r e p a r a t i o n (1 g o f m i t o c h o n -d r i a l a c e t o n e powder e x t r a c t e d w i t h 10 m l o f a 0.154 M KC1 s o l u -t i o n ) ; 5.5 y m o l e s o f NADPH d i s s o l v e d i n 0.5 m l o f T r i s - M g C l 2 b u f f e r . The t o t a l v o l u m e was 5.1 m l . A f t e r 1 5 , 30, 45, 60 o r 90 m i n u t e s o f i n c u b a t i o n , r e a c t i o n s w e r e t e r m i n a t e d by t h e a d -d i t i o n o f e t h y l a c e t a t e (15 ml) t o e a c h f l a s k . - 47 -t o 1 1 6 - h y d r o x y l a t i o n , i . e . , t h e i n c r e a s e i n NADPH o x i d a t i o n p r o d u c e d by t h e a d d i t i o n o f d e o x y c o r t i c o s t e r o n e ( F i g . 4 ) , and a c t u a l p r o d u c t i o n o f c o r t i c o s t e r o n e ( T a b l e I I ) was made. A f t e r 1 5 , 30, and 45 m i n u t e s o f i n c u b a t i o n , t h e amount o f c o r t i c o s t e r o n e p r o d u c e d was 0.25, 0.38, a n d 0.52 y m o l e ^ r e s p e c t i v e l y . The i n c r e a s e d amount o f NADPH o x i d i z e d u p o n a d d i t i o n o f d e o x y c o r t i c o s t e r o n e was 0.63, 0.90, and 1.3 u m o l e s a f t e r t h e s e same i n c u b a t i o n p e r i o d s . Thus t h e r a t i o o f NADPH o x i d i z e d t o c o r t i c o s t e r o n e p r o d u c e d was 2.5, 2.4, and 2.9 a f t e r 15, 30, and 45 m i n u t e s o f i n c u b a t i o n . The s t o i c h i o m e t r y o f t h i s 1 1 3 - h y d r o x y l a t i o n r e a c t i o n d i f f e r s f r o m t h a t o f t h e m i c r o s o m a l s t e r o i d 2 1 - h y d r o x y l a s e . C o o p e r et al. (40) showed t h a t i n t h e h y d r o x y l a t i o n o f s t e r o i d s a t C - 2 1 , one m o l e o f NADPH was o x i d i z e d f o r e a c h m o l e o f C - 2 1 h y d r o x y l a t e d s t e r o i d f o r m e d . I n o r d e r t o a s c e r t a i n t h e r e l a t i o n s h i p b e t w e e n NADPH o x i d a t i o n a n d 1 1 3 - h y d r o x y l a t i o n , t h e e f f e c t s o f some known e l e c t r o n t r a n s p o r t i n h i b i t o r s on t h e s e p r o c e s s e s w e r e e x a m i n e d . The d a t a i n T a b l e I I I i l l u s t r a t e s t h e e f f e c t s o f t h e s e i n -h i b i t o r s o n NADPH o x i d a t i o n i n t h e a b s e n c e o f s t e r o i d s u b -s t r a t e . P o t a s s i u m c y a n i d e h a s no e f f e c t on t h e o x i d a t i o n o f NADPH. T h i s compound i s a w e l l - e s t a b l i s h e d i n h i b i t o r o f c y t o c h r o m e o x i d a s e , t h e . t e r m i n a l o x i d a s e o f t h e e l e c t r o n t r a n s p o r t c h a i n . L a c k o f i n h i b i t i o n o f NADPH o x i d a t i o n by p o t a s s i u m c y a n i d e s u g g e s t s t h a t o x i d a t i o n o f NADPH i s n o t p r o c e e d i n g v i a t h e c l a s s i c a l e l e c t r o n t r a n s p o r t c h a i n . M o r e o v e r , c y t o c h r o m e o x i d a s e i s r e a d i l y d e s t r o y e d b y . a c e t o n e - 48 -T A B L E I I I E f f e c t o f i n h i b i t o r s o n NADPH o x i d a t i o n I n c u b a t i o n t i m e ( m i n u t e s ) P e r c e n t o f c o n t r o l a c t i v i t y I n h i b i t o r a d d e d KCN A n t i m y c i n A A m y t a l D i c u m a r o l 5 104 100 96 118 10 100 89 94 124 15 100 91 94 131 20 100 89 93 136 A p p r o p r i a t e r e a c t i o n f l a s k s c o n t a i n e d : 1 .0 m l o f T r i s MgCl2 b u f f e r ; 2 m l o f enzyme p r e p a r a t i o n (1 g o f m i t o c h o n d r i a l a c e t o n e powder e x t r a c t e d w i t h 15 m l o f a 0 .154 M K C 1 s o l u t i o n ) ; 0 . 1 m l o f a 3 . 0 x 1 0 - 2 M r s o l u t i o n o f p o t a s s i u m c y a n i d e , f i n a l c o n c e n t r a t i o n , 9 . 5 x 1 0 ~ 3 ' m o l e s / 1 ; 0 . 0 4 m l o f a 1 .8 x 10~3 M s o l u t i o n o f a n t i m y c i n A , f i n a l c o n c e n t r a t i o n , 2 . 3 x 1 0 - 5 m o l e s / 1; 0 . 1 m l o f a 2 . 0 x 1 0 " 2 M s o l u t i o n o f a m y t a l , f i n a l c o n c e n -t r a t i o n , 6 . 4 x 1 0 ~ 4 m o l e s / 1 ; o r 0.0 5 m l o f a 1 .0 x 1 0 ~ 2 M s o l u -t i o n o f d i c u m a r o l , f i n a l c o n c e n t r a t i o n , 1 .6 x 1 0 - 4 m o l e s / 1 . T h e v o l u m e o f e a c h r e a c t i o n m i x t u r e was a d j u s t e d t o 3 . 1 5 m l w i t h w a t e r a n d 1 .0 y m o l e o f NADPH d i s s o l v e d i n 0 . 0 5 m l o f T r i s -MgCl2 b u f f e r was a d d e d a t z e r o t i m e . - 49 -t r e a t m e n t (31,121) a n d i s u n l i k e l y t o be p r e s e n t i n a n a c e t o n e powder o f b e e f a d r e n a l m i t o c h o n d r i a . Two o t h e r i n h i b i t o r s t e s t e d , a m y t a l a n d a n t i m y c i n A h a v e no e f f e c t o n NADPH o x i d a -t i o n , c o n f i r m i n g t h a t t h e e l e c t r o n t r a n s p o r t c h a i n i s n o t f u n c t i o n i n g i n t h i s s y s t e m . D i c u m a r o l , a n u n c o u p l e r o f o x i -d a t i v e p h o s p h o r y l a t i o n (122-124) , p r o d u c e s a s l i g h t i n c r e a s e i n t h e r a t e o f NADPH o x i d a t i o n . I n c r e a s e d r e s p i r a t i o n by m i t o c h o n d r i a i n t h e p r e s e n c e o f d i c u m a r o l , a s w e l l a s o t h e r u n c o u p l i n g a g e n t s h a s b e e n r e p o r t e d (39,125). W h e t h e r t h e i n c r e a s e i n NADPH o x i d a t i o n d e m o n s t r a t e d i n T a b l e I I I i s r e l a t e d t o t h e s t i m u l a t i o n o f r e s p i r a t i o n r e p o r t e d i n s t u d i e s w i t h i n t a c t m i t o c h o n d r i a i s u n c e r t a i n . A l t e r n a t i v e l y , d i c u m a r o l may be f u n c t i o n i n g a s a n e l e c t r o n a c c e p t o r i n t h i s s y s t e m . E x a m i n a t i o n o f t h e m o l e c u l a r s t r u c t u r e o f d i c u m a r o l ( F i g . 3) i n d i c a t e s p o s i t i o n s t h a t c o u l d p o s s i b l y u n d e r g o r e -d u c t i o n o r h y d r o x y l a t i o n . The e f f e c t s o f t h e s e i n h i b i t o r s o h 1 1 6 - h y d r o x y l a t i o n a r e shown i n T a b l e I V . N e i t h e r a m y t a l n o r a n t i m y c i n A p r o d u c e s any i n h i b i t i o n o f 1 1 6 - h y d r o x y l a t i o n . P o t a s s i u m c y a n i d e p r o d u c e s a s l i g h t i n h i b i t i o n . S t u d i e s b y s e v e r a l g r o u p s (70,115,117,126) h a v e shown t h a t N A D P H - s u p p o r t e d 1 1 6 - h y d r o x y l a t i o n i s g e n e r a l l y i n s e n s i t i v e t o c y a n i d e , a l t h o u g h F o n z o et al. (126) d i d f i n d s l i g h t i n h i b i t i o n p r o d u c e d a t h i g h e r c o n c e n t r a t i o n s o f c y a n i d e (2-4 mM) when t h e c o n c e n t r a t i o n o f NADPH became r a t e - l i m i t i n g . Schenkman et al. (97) h a v e o b s e r v e d t h a t p o t a s s i u m c y a n i d e c o u l d b i n d t o t h e m i c r o s o m a l h y d r o x y l a s e o f r a t l i v e r . The c o n c e n t r a -t i o n a t w h i c h p o t a s s i u m c y a n i d e . i n t e r a c t e d w i t h t h e m i c r o s o m a l h y -d r o x y l a s e was c o n s i d e r a b l y h i g h e r t h a n t h e s u b s t r a t e c o n c e n t r a t i o n - 50 -T A B L E I V E f f e c t o f i n h i b i t o r s o n t h e 1 1 3 - h y d r o x y l a t i o n o f d e o x y c o r t i -c o s t e r o n e I n h i b i t o r a d d e d F i n a l c o n c e n t r a t i o n P e r c e n t o f c o n t r o l ( m o l e s / 1 ) a c t i v i t y KCN 1 .0 X I O " 3 77 A n t i m y c i n A 2 . 5 X I O " 5 -4 98 A m y t a l 6 .6 X 10 100 D i c u m a r o l -, „ - 4 3 . 2 X 10 , - 4 0 D i c u m a r o l 1 .9 X 10 11 D i c u m a r o l , - 5 43 9 . 5 X 10 A p p r o p r i a t e r e a c t i o n f l a s k s c o n t a i n e d : 0 . 7 6 u m o l e o f d e o x y c o r t i c o s t e r o n e d i s s o l v e d i n 0 . 0 5 m l o f p r o p y l e n e g l y c o l ; 1 .50 m l o f T r i s-MgCl2 b u f f e r ; 3 m l o f enzyme s o l u t i o n (1 g o f m i t o c h o n d r i a l a c e t o n e powder e x t r a c t e d w i t h 15 m l o f a 0 . 1 5 4 M K C l s o l u t i o n ) ; i n h i b i t o r s o l u t i o n s a s d e s c r i b e d i n T a b l e I I I t o g i v e t h e f i n a l c o n c e n t r a t i o n i n d i c a t e d i n t h e t a b l e . T h e v o l u m e o f e a c h r e a c t i o n m i x t u r e was a d j u s t e d t o 5 . 0 m l w i t h w a t e r a n d 4 u m o l e s o f NADPH d i s s o l v e d i n 0 .2 m l o f T r i s-MgCl2 b u f f e r was a d d e d a t z e r o t i m e . A f t e r 60 m i n u t e s o f i n c u b a t i o n , r e a c t i o n s w e r e t e r m i n a t e d by t h e a d d i t i o n o f e t h y l a c e t a t e (15 ml ) t o e a c h f l a s k . - 51 -r e q u i r e d f o r a s i m i l a r i n t e r a c t i o n w i t h t h i s s y s t e m . T h e s l i g h t i n h i b i t i o n o f 1 1 3 - h y d r o x y l a t i o n p r o d u c e d by c y a n i d e u n d e r t h e p r e s e n t e x p e r i m e n t a l c o n d i t i o n s ( T a b l e IV) s u g g e s t s t h a t ' p o t a s s i u m c y a n i d e c a n i n t e r a c t t o a l i m i t e d e x t e n t w i t h t h e 113 - h y d r o x y l a s e . T h e d a t a i n T a b l e I V show t h a t d i c u m a r o l i n h i b i t s t h e 1 1 3 - h y d r o x y l a t i o n o f d e o x y c o r t i c o s t e r o n e . One h u n d r e d p e r -c e n t i n h i b i t i o n o f c o r t i c o s t e r o n e f o r m a t i o n i s a c h i e v e d a t - 4 a d x c u m a r o l c o n c e n t r a t i o n o f 3 . 2 x 10 m o l e s/1. T h i s i n -h i b i t i o n i s o b t a i n e d i n s p i t e o f t h e f a c t t h a t n e i t h e r t h e e l e c t r o n t r a n s p o r t c h a i n n o r h i g h e n e r g y compounds i n t h e f o r m o f A T P a r e r e q u i r e d f o r 1 1 3 - h y d r o x y l a t i o n when NADPH i s e m p l o y e d as t h e c o f a c t o r . I t i s a p p a r e n t t h a t t h e i n h i b i -t i o n o f 1 1 3 - h y d r o x y l a t i o n by d i c u m a r o l u n d e r t h e s e c o n d i -t i o n s i s n o t a f u n c t i o n o f i t s a b i l i t y t o u n c o u p l e o x i d a -t i v e p h o s p h o r y l a t i o n , a n d t h a t a n a l t e r n a t e m e c h a n i s m i s i n -v o l v e d . (b) K i n e t i c S t u d y o f 1 1 3 - H y d r o x y l a t i o n E x p e r i m e n t s t o e s t a b l i s h t h e k i n e t i c s o f 113 - h y d r o x y -l a t i o n by t h e enzyme s y s t e m e x t r a c t e d f r o m t h e a c e t o n e p o w d e r o f b e e f a d r e n a l m i t o c h o n d r i a w e r e c a r r i e d o u t . T h e f o r m a -t i o n o f c o r t i c o s t e r o n e a s a f u n c t i o n o f i n c u b a t i o n t i m e a t two s u b s t r a t e c o n c e n t r a t i o n s i s shown i n F i g u r e 5. A t t h e h i g h e r s u b s t r a t e c o n c e n t r a t i o n (22 umoles/1) t h e r a t e o f 1 1 3 - h y d r o x y l a t i o n i s c o n s t a n t d u r i n g t h e f i r s t s i x m i n u t e s o f i n c u b a t i o n . A t t h i s p o i n t 17% o f t h e s u b s t r a t e d e o x y c o r t i c o -F i g . 5. The r a t e o f 1 1 6 - h y d r o x y l a t i o n a s a f u n c t i o n o f d e o x y c o r t i c o s t e r o n e c o n c e n t r a t i o n . E a c h r e a c t i o n f l a s k c o n t a i n e d : d e o x y c o r t i c o s t e r o n e - 1 , 2- 3H ( s p e c i f i c a c t i v i t y 2.15 x 1 0 7 cpm/umole) d i s s o l v e d i n 0.05 ml o f p r o p y l e n e g l y c o l ; 1.35 m l o f T r i s - M g C l 2 b u f f e r ; 2.6 m l o f enzyme p r e p a r a t i o n ; and 0.1 m l o f a d i l u t e a l c o -h o l i c KOH s o l u t i o n . R e a c t i o n m i x t u r e s w e r e p r e - i n c u b a t e d f o r 5 m i n u t e s . NADPH (3.3 u m o l e s ) d i s s o l v e d i n 0.2 m l o f T r i s - M g C l 2 b u f f e r was a d d e d t o e a c h r e a c t i o n f l a s k a t z e r o t i m e . 1.0 m l s a m p l e s w e r e r e m o v e d f r o m e a c h r e a c t i o n f l a s k a f t e r 3, 6, 9, a n d 12 m i n u t e s o f i n c u b a t i o n a n d ' p i p e t t e d i n t o e t h y l a c e t a t e (2 ml) t o t e r m i n a t e t h e r e a c t i o n s . D e o x y c o r t i c o s t e r o n e f i n a l c o n c e n t r a t i o n , u m o l e s / 1 : 4.4 (©); 22.2 (O). - 53 -s t e r o n e h a s b e e n c o n v e r t e d t o c o r t i c o s t e r o n e . A t t h e l o w e r s u b s t r a t e c o n c e n t r a t i o n ( 4 . 4 u m o l e s / 1 ) t h e r a t e o f 1 1 3 - h y d r o x y -l a t i o n r a p i d l y becomes n o n l i n e a r , d u e t o u t i l i z a t i o n o f s t e r o i d s u b s t r a t e . A t t h i s c o n c e n t r a t i o n , s t e r o i d s u b s t r a t e v e r y q u i c k l y becomes t h e r a t e - l i m i t i n g f a c t o r i n 1 1 3 - h y d r o x y l a t i o n . A p l o t o f t h e i n i t i a l v e l o c i t y o f 1 1 3 - h y d r o x y l a t i o n a g a i n s t s u b s t r a t e c o n c e n t r a t i o n ( F i g . 6) p r o d u c e s a t y p i c a l h y p e r -b o l i c s a t u r a t i o n c u r v e . K i n e t i c p r e s e n t a t i o n o f t h i s d a t a ( F i g . 6) i n d i c a t e s t h a t t h e K m f o r 1 1 3 - h y d r o x y l a t i o n o f d e o x y -c o r t i c o s t e r o n e i s 5 . 5 u m o l e s / 1 . T h i s v a l u e i s i n c l o s e : a g r e e m e n t w i t h t h a t d e t e r m i n e d by Sharma et al. ( 3 3 ) . T h e y o b t a i n e d a K m f o r d e o x y c o r t i c o s t e r o n e o f 8 . 5 u m o l e s / 1 e m p l o y -i n g a l y o p h i l i z e d enzyme p r e p a r a t i o n o b t a i n e d f r o m a 1 0 5 , 0 00 x g s u p e r n a t a n t o f s o n i c a t e d b e e f a d r e n a l m i t o c h o n d r i a . How-e v e r , i t i s l o w e r t h a n t h e K o f 20 u m o l e s / 1 o b t a i n e d by m J D o m i n g u e z a n d S a m u e l s (45) e m p l o y i n g a w h o l e h o m o g e n a t e o f r a t a d r e n a l g l a n d s . T h i s d i s c r e p a n c y may b e d u e t o t h e d i f f e r e n t e n z y m e ? p r e p a r a t i o n s e m p l o y e d ; t h a t i s a m i t o c h o n -d r i a l e x t r a c t v s . a n a d r e n a l h o m o g e n a t e . (c) S p e c t r o p h o t o m e t r y S t u d i e s o n t h e Enzyme P r e p a r a t i o n S p e c t r o p h o t o m e t r i c e x a m i n a t i o n o f a d r e n a l m i t o c h o n d r i a h a s shown t h a t t h e m i t o c h o n d r i a o f t h i s t i s s u e p o s s e s s t h e c o m p o n e n t s o f t h e c l a s s i c a l r e s p i r a t o r y c h a i n ( 3 9 , 1 1 7 , 1 2 7 ) . I n a d d i t i o n , t h e r e i s p r e s e n t i n t h e s e m i t o c h o n d r i a a s e c o n d t y p e o f r e s p i r a t o r y c h a i n t h a t t r a n s f e r s e l e c t r o n s f r o m NADPH t o c y t o c h r o m e P - 4 50 f o r t h e 1 1 3 - h y d r o x y l a t i o n o f - 54 -[S] n.rnoles/1 F i g . 6 . [S]/v versus [S] p l o t f o r the 113-hydroxylation of deoxycorticosterone. Each r e a c t i o n f l a s k contained: d e o x y c o r t i c o s t e r o n e - 1 , 2- 3H ( s p e c i f i c a c t i v i t y 6.24 x 10 7 cpm/umole) d i s s o l v e d i n 0.05 ml of propylene g l y c o l ; 0.70 ml of T r i s - M g C l 2 b u f f e r ; 1.30 ml of enzyme p r e p a r a t i o n ; and 0.05 ml of a d i l u t e a l c o -h o l i c KOH s o l u t i o n . Reaction mixtures were pre-incubated f o r 8 minutes. NADPH (1.6 umoles) d i s s o l v e d i n 0.05 ml of T r i s - M g C l 2 b u f f e r was added to each f l a s k at zero time. A f t e r 90 seconds of i n c u b a t i o n a sample (1.0 ml) was removed from each f l a s k and p i p e t t e d i n t o e t h y l acetate (2 ml) to termin-ate the r e a c t i o n . • - 55 -d e o x y c o r t i c o s t e r o n e ( 3 9 , 5 6 , 1 2 7 ) . S p e c t r o p h o t o m e t r i e e x a m i n a -t i o n o f t h e enzyme p r e p a r a t i o n ( e x t r a c t e d f r o m a n a c e t o n e powder o f b o v i n e a d r e n a l m i t o c h o n d r i a ) e m p l o y e d i n o u r s t u d i e s was t h e r e f o r e u n d e r t a k e n . ( i ) R e d u c e d - o x i d i z e d a n d c a r b o n m o n o x i d e d i f f e r e n c e s p e c t r a o f t h e enzyme p r e p a r a t i o n . The r e d u c e d m i n u s o x i d i z e d d i f f e r e n c e s p e c t r u m o f t h e enzyme p r e p a r a t i o n i s shown i n F i g u r e 7 . T h e s e s p e c t r a a r e p r o d u c e d b o t h by e n z y m a t i c r e d u c t i o n w i t h NADPH a n d b y c h e m i c a l r e d u c t i o n w i t h s o d i u m d i t h i o n i t e (Na2S20i+) . T h e d i f f e r e n c e s p e c t r u m p r o d u c e d by r e d u c t i o n w i t h NADPH e x -h i b i t s a b s o r p t i o n maxima a t 5 7 0 , 5 4 5 , a n d 419 mu a n d a t r o u g h a t 460 - 470 my . T h e a b s o r p t i o n maximum a t 419 my i s s i m i l a r t o t h a t o f c y t o c h r o m e c ( 8 5 ) , b u t t h e maxima a t 570 a n d 545 my do n o t c o r r e s p o n d t o t h e a b s o r p t i o n maxima e x h i b i t e d b y a n y o f t h e n o r m a l r e s p i r a t o r y c h a i n c y t o c h r o m e s i n t h i s r e g i o n o f t h e s p e c t r u m ( 8 5 , 1 2 7 ) . The f o r m a t i o n o f t h e a b s o r p t i o n maximum a t 419 my u p o n a d d i t i o n o f NADPH may i n d i c a t e t h e p r e s e n c e o f a N A D P H - c y t o c h r o m e c r e d u c t a s e i n t h e enzyme p r e p a r a t i o n . No a b s o r p t i o n maxima c o r r e s p o n d i n g c o r r e s p o n d i n g t o c y t o c h r o m e s a o r as, c y t o c h r o m e b, o r c y t o c h r o m e P - 4 2 0 a r e o b s e r v e d . A s m e n t i o n e d p r e v i o u s l y , i t i s q u i t e l i k e l y t h a t c y t o c h r o m e s a a n d a 3 a r e d e s t r o y e d d u r i n g p r e p a r a t i o n o f t h e a c e t o n e p o w d e r . T h e d e c r e a s e i n a b s o r b a n c e i n t h e r e g i o n 460 - 470 my s u g g e s t s t h e r e d u c t i o n o f a f l a v o p r o t e i n p r e s e n t i n t h e enzyme p r e p a r a t i o n ( 8 5 ) . - 56 -0.16 0 . 0 6 0.04 -0 . 0 2 o < CD a: o m < •0.0 2 -- 0 . 0 4 -0 . 0 6 4 0 0 4 5 0 5 0 0 5 5 0 WAVELENGTH,m/x 6 0 0 F i g . 7 . Reduced minus o x i d i z e d d i f f e r e n c e spectrum o f the mito-c h o n d r i a l acetone powder enzyme p r e p a r a t i o n reduced w i t h NADPH or sodium d i t h i o n i t e . Each c u v e t t e c o n t a i n e d : 0.05 ml of p r o p y l e n e g l y c o l ; 1.0 ml of T r i s - M g C l 2 b u f f e r ; and 1.95 ml of enzyme p r e p a r a t i o n . NADPH (1.1 y m o l e s ) , d i s s o l v e d i n 0.05 ml of T r i s - M g C l 2 , o r sodium d i t h i o n i t e was added to the sample c u v e t t e and the d i f f e r e n c e spectrum was r e c o r d e d . NADPH reduced d i f f e r e n c e spectrum ( ) ; d i t h i o n i t e r e d u c e d d i f f e r e n c e spectrum ( ) . - 57 -C h e m i c a l r e d u c t i o n o f t h e enzyme p r e p a r a t i o n w i t h s o d i u m d i t h i o n i t e p r o d u c e s a d i f f e r e n c e s p e c t r u m w i t h a b s o r p t i o n maxima a t 5 5 6 , 5 1 8 , a n d 430 my a n d w i t h a t r o u g h a t 460 my. T h e maxima p r e s e n t i n t h e N A D P H - r e d u c e d d i f f e r e n c e s p e c t r u m a r e no l o n g e r e v i d e n t , b e i n g o b s c u r e d by t h e more p r o m i n e n t c h e m i c a l l y p r o d u c e d m a x i m a . T h e d i t h i o n i t e - p r o d u c e d a b s o r p -t i o n maximum a t 430 my h a s t w i c e t h e i n t e n s i t y o f t h e N A D P H -p r o d u c e d a b s o r p t i o n maximum a t 419 my. T h e d i t h i o n i t e - p r o -d u c e d d i f f e r e n c e s p e c t r u m i s i d e n t i c a l t o t h e d i f f e r e n c e s p e c t r u m o f h e m o g l o b i n p r o d u c e d by d i t h i o n i t e a n d m o s t p r o b a b l y r e p r e s e n t s h e m o g l o b i n c o n t a m i n a t i o n o f t h e enzyme p r e p a r a t i o n ( 8 4 , 1 1 7 , 1 2 7 ) . C y t o c h r o m e b may a l s o be c o n -t r i b u t i n g t o t h i s d i t h i o n i t e - p r o d u c e d d i f f e r e n c e s p e c t r u m ( 8 5 , 1 2 7 ) . T h e c a r b o n m o n o x i d e d i f f e r e n c e s p e c t r a o f t h e enzyme p r e p a r a t i o n r e d u c e d w i t h b o t h NADPH a n d d i t h i o n i t e a r e shown i n F i g u r e 8 . T h e two s p e c t r a a r e i d e n t i c a l w i t h a b -s o r p t i o n maxima a t 4 7 0 , 5 3 4 , 457 - 460 a n d 418 my, a n d c o r r e s p o n d t o t h e c a r b o n m o n o x i d e d i f f e r e n c e s p e c t r u m o f a h e m o g l o b i n s o l u t i o n t r e a t e d i n t h e same m a n n e r . T h e r e i s no a b s o r p t i o n maximum a t 4 50 my c o r r e s p o n d i n g t o c y t o c h r o m e P - 4 5 0 . T h i s a b s o r p t i o n b a n d h a s b e e n shown t o be a d i s -t i n c t f e a t u r e o f t h e c a r b o n m o n o x i d e d i f f e r e n c e s p e c t r u m o f a d r e n a l m i t o c h o n d r i a ( 5 6 , 7 0 , 8 5 , 8 6 , 1 2 7 ) . I n a d d i t i o n no a b s o r p t i o n maxima c o r r e s p o n d i n g t o t h e c a r b o n m o n o x i d e c o m p l e x o f c y t o c h r o m e s a a n d a 3 a r e o b s e r v e d . - 58 -UJ (J < CO a o (/) m < 400 450 500 550 WAVELENGTH, muu 600 F i g . 8 . C a r b o n m o n o x i d e d i f f e r e n c e s p e c t r u m o f t h e m i t o c h o n -d r i a l a c e t o n e p o w d e r enzyme p r e p a r a t i o n r e d u c e d w i t h NADPH o r s o d i u m d i t h i o n i t e . I n c u b a t i o n s 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 i n F i g . 7 . NADPH ( 1 . 1 u m o l e s ) d i s s o l v e d i n 0 . 0 5 m l T r i s - M g C l 2 r o r s o d i u m d i t h i o n i t e was a d d e d t o b o t h t h e s a m p l e a n d r e f e r e n c e c u v e t t e s . C a r b o n m o n o x i d e was b u b b l e d t h r o u g h t h e s a m p l e c u v e t t e s o l u -t i o n f o r 60 s e c o n d s a n d t h e d i f f e r e n c e s p e c t r u m was r e c o r d e d . NADPH r e d u c e d ( ) ; s o d i u m d i t h i o n i t e r e d u c e d ( ) . - 59 -( i i ) Ammonium s u l f a t e f r a c t i o n a t i o n o f t h e enzyme p r e p a r -a t i o n . B e c a u s e o f t h e i n t e r f e r e n c e b y h e m o g l o b i n i n a t t e m p t s t o i d e n t i f y c y t o c h r o m e P-450 as a component o f t h e enzyme p r e -p a r a t i o n e m p l o y e d i n t h e p r e s e n t s t u d i e s , t h e ammonium s u l -f a t e f r a c t i o n a t i o n p r o c e d u r e o f K i m u r a a n d S u z u k i (59) was e m p l o y e d i n o r d e r t o s e p a r a t e a n y c y t o c h r o m e P-4 50 t h a t may be p r e s e n t i n t h e enzyme p r e p a r a t i o n f r o m h e m o g l o b i n . By t h i s p r o c e d u r e c y t o c h r o m e s P-450 a n d P-420 c a n be o b t a i n e d i n a f r a c t i o n p r e c i p i t a t i n g b e t w e e n 20% and 40% s a t u r a t i o n w i t h ammonium s u l f a t e . A s i m i l a r p r o c e d u r e h a s b e e n r e -p o r t e d b y Sweat a n d Young ( 1 2 8 ) . The r e d u c e d m i n u s o x i d i z e d d i f f e r e n c e s p e c t r u m a n d t h e c a r b o n m o n o x i d e d i f f e r e n c e s p e c t r u m o f t h e f r a c t i o n s p r e c i p i t a t i n g b e t w e e n 0% a n d 2 0 % , 20% and 4 0 % , and 40% a n d 80% s a t u r a t i o n w i t h ammonium s u l f a t e a r e shown i n F i g u r e s 9, 10, a n d 11 r e s p e c t i v e l y . B o t h t h e r e d u c e d m i n u s o x i d i z e d d i f f e r e n c e s p e c t r u m ( F i g . 9a) a n d t h e c a r b o n m o n o x i d e d i f -f e r e n c e s p e c t r u m ( F i g . 9b) o f t h e f r a c t i o n p r e c i p i t a t i n g b e t w e e n 0% and 20% s a t u r a t i o n w i t h ammonium s u l f a t e a r e i d e n -t i c a l t o t h o s e o b t a i n e d w i t h h e m o g l o b i n . The r e d u c e d m i n u s o x i d i z e d d i f f e r e n c e : s p e c t r u m o f t h e f r a c t i o n p r e c i p i t a t i n g b e t w e e n 20% a n d 40% s a t u r a t i o n w i t h ammonium s u l f a t e e x h i b i t s a b s o r p t i o n maxima a t 555 - 557, 5 3 2 , a n d 427 my ( F i g . 1 0 a ) . T h e s e a b s o r p t i o n maxima c o r r e s p o n d c l o s e l y t o t h o s e e x h i b i t e d i n t h e d i f f e r e n c e s p e c t r u m o f c y t o c h r o m e P-4 20 o f r a b b i t l i v e r m i c r o s o m e s r e p o r t e d b y Omura 4 0 0 4 5 0 5 0 0 5 5 0 6 0 0 4 0 0 4 5 0 5 0 0 5 5 0 6 0 0 F i g . 9a W A V E L E N G T H , mjx F i g . 9 b F i g . 9 a . D i t h i o n i t e r e d u c e d m i n u s o x i d i z e d d i f f e r e n c e s p e c t r u m o f t h e 0-20% ammonium s u l -f a t e f r a c t i o n o f t h e m i t o c h o n d r i a l a c e t o n e p o w d e r enzyme p r e p a r a t i o n . T h e 0-20% ammonium s u l f a t e f r a c t i o n o b t a i n e d f r o m 25 m l o f enzyme p r e p a r a t i o n (1 g m i t o c h o n d r i a l a c e t o n e p o w d e r e x t r a c t e d w i t h 25 m l o f a 0 . 1 5 4 M K C l s o l u t i o n ) was d i s s o l v e d i n 1 .8 m l o f a s o l u t i o n made up o f T r i s - M g C l 2 b u f f e r ( 0 . 6 ml) a n d 0 . 1 5 4 M K C l ( 1 . 2 ml) and d i v i d e d e q u a l l y b e t w e e n two c u v e t t e s . A few c r y s t a l s o f s o d i u m d i t h i o n i t e w e r e a d d e d t o t h e s a m p l e c u v e t t e a n d t h e d i f f e r e n c e s p e c t r u m was r e c o r d e d . F i g . 9 b . D i t h i o n i t e r e d u c e d c a r b o n m o n o x i d e d i f f e r e n c e s p e c t r u m o f t h e 0-20% ammonium s u l f a t e f r a c t i o n o f t h e m i t o c h o n d r i a l a c e t o n e p o w d e r enzyme p r e p a r a t i o n . S o d i u m d i t h i o n i t e a l s o was a d d e d t o t h e r e f e r e n c e c u v e t t e o f 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 . 9 a . C a r b o n m o n o x i d e was b u b b l e d t h r o u g h t h e s a m p l e c u v e t t e s o l u t i o n f o r 60 s e c o n d s a n d t h e d i f f e r e n c e s p e c t r u m was r e c o r d e d . - 61 -427 J 1 L 400 450 500 550 600 400 450 500 550 600 WAVELENGTH.mn F i g . 10a F i g . 10b F i g . 1 0 a . D i t h i o n i t e r e d u c e d m i n u s o x i d i z e d d i f f e r e n c e s p e c t r u m o f t h e 20-40% ammonium s u l f a t e f r a c t i o n o f t h e m i t o -c h o n d r i a l a c e t o n e p o w d e r enzyme p r e p a r a t i o n . T h e d i f f e r e n c e s p e c t r u m was d e t e r m i n e d as d e s c r i b e d i n F i g . 9 a . T h e p r e c i p i t a t e o b t a i n e d b e t w e e n 20% a n d 40% ammonium s u l f a t e s a t u r a t i o n was d i s s o l v e d i n 3 . 0 m l o f t h e T r i s - K C l b u f f e r m i x t u r e . F i g . 1 0 b . D i t h i o n i t e r e d u c e d c a r b o n m o n o x i d e d i f f e r e n c e s p e c -t r u m o f t h e 20-40% ammonium s u l f a t e f r a c t i o n o f t h e m i t o c h o n d r i a l a c e t o n e p o w d e r enzyme p r e p a r a t i o n . T h e d i f f e r e n c e s p e c t r u m was d e t e r m i n e d as d e s c r i b e d i n F i g . 9 b . 400 450 500 550 6 0 0 4 0 0 450 500 550 6 0 0 F i g . 11a WAVELENGTH, m/x F i g . ' l l b F i g . 1 1 a . D i t h i o n i t e r e d u c e d m i n u s o x i d i z e d d i f f e r e n c e s p e c t r u m o f t h e 40-80% ammonium s u l f a t e f r a c t i o n o f t h e m i t o c h o n d r i a l a c e t o n e p o w d e r enzyme p r e p a r a t i o n . T h e d i f f e r e n c e s p e c t r u m was d e t e r m i n e d a s d e s c r i b e d - i n F i g . 9 a . T h e p r e c i p i t a t e o b -t a i n e d b e t w e e n 40% a n d 80% s a t u r a t i o n w i t h ammonium s u l f a t e was d i s s o l v e d i n 6 . 0 m l o f t h T r i s - K C l b u f f e r m i x t u r e . F i g . l i b . D i t h i o n i t e r e d u c e d c a r b o n m o n o x i d e d i f f e r e n c e s p e c t r u m o f t h e 40-80% ammonium s u l f a t e f r a c t i o n o f t h e m i t o c h o n d r i a l a c e t o n e p o w d e r enzyme p r e p a r a t i o n . T h e d i f f e r e n c e s p e c t r u m was d e t e r m i n e d a s d e s c r i b e d i n F i g . 9 b . - 63 -a n d S a t o ( 8 8 , 8 9 ) . i t i s a l s o p o s s i b l e t h a t h e m o g l o b i n i s c o n t r i b u t i n g t o t h i s s p e c t r u m . The a b s o r p t i o n maxima e x -h i b i t e d by c y t o c h r o m e P - 4 2 0 a n d h e m o g l o b i n a r e q u i t e s i m i -l a r a l t h o u g h t h e s o r e t b a n d f o r c y t o c h r o m e P - 4 2 0 i s a t 427 my w h i l e t h a t f o r h e m o g l o b i n i s a t 430 my. A l s o p r e s e n t i n t h e r e d u c e d m i n u s o x i d i z e d d i f f e r e n c e s p e c t r u m o f t h e f r a c t i o n p r e c i p i t a t i n g b e t w e e n 20% a n d 40% s a t u r a t i o n w i t h ammonium s u l f a t e i s a t r o u g h w i t h a maximum a t 456 my. T h i s t r o u g h i s p r o d u c e d b y r e d u c t i o n o f f l a v o p r o t e i n a n d may r e p r e s e n t t h e NADPH r e d u c t a s e ( a d r e n o d o x i n r e d u c t a s e ) t h a t i s a c o m -p o n e n t o f t h e l l p - h y d r o x y l a s e p a t h w a y ( 5 6 , 5 7 ) . K i m u r a a n d S u z u k i (59) h a v e shown t h a t t h i s f l a v o p r o t e i n p r e c i p i t a t e s b e t w e e n 20% a n d 40% s a t u r a t i o n w i t h ammonium s u l f a t e . T h e c a r b o n m o n o x i d e d i f f e r e n c e s p e c t r u m o f t h e f r a c t i o n p r e c i p i t a t i n g b e t w e e n 20% a n d 40% s a t u r a t i o n w i t h . a m m o n i u m s u l f a t e shows a n i n t e n s e a b s o r p t i o n b a n d a t 450 my i n d i c a t i n g t h e p r e s e n c e o f c y t o c h r o m e P - 4 5 0 i n t h i s f r a c t i o n ( F i g . 1 0 b ) . A l s o p r e s e n t a r e a b s o r p t i o n maxima a t 5 7 2 , 5 3 6 , a n d 418 my, w h i c h may r e p r e s e n t c y t o c h r o m e P - 4 2 0 , a l t h o u g h t h e s e maxima a r e a t w a v e l e n g t h s s l i g h t l y s h o r t e r t h a n t h o s e r e p o r t e d by Omura a n d S a t o ( 8 8 , 8 9 ) f o r c y t o c h r o m e P - 4 2 0 o f r a b b i t l i v e r m i c r o s o m e s . A g a i n h e m o g l o b i n may b e c o n t r i b u t i n g t o t h i s a b s o r p t i o n s p e c t r u m . C y t o c h r o m e P - 4 2 0 i s n o t a n o r m a l c o n -s t i t u e n t o f t h e a d r e n a l m i t o c h o n d r i a , b u t i s p r o d u c e d by d e -c o m p o s i t i o n o f c y t o c h r o m e P - 4 5 0 ( 8 8 - 9 3 ) . O u r e x p e r i m e n t s i n d i c a t e t h a t c y t o c h r o m e P - 4 2 0 i s p r e s e n t i n t h e a c e t o n e pow-d e r o f a d r e n a l m i t o c h o n d r i a . - 6.4 -T h e r e d u c e d m i n u s o x i d i z e d d i f f e r e n c e s p e c t r u m o f t h e f r a c t i o n p r e c i p i t a t i n g b e t w e e n 40% a n d 80% s a t u r a t i o n w i t h ammonium s u l f a t e e x h i b i t s a b s o r p t i o n maxima a t 5 9 2 , 5 5 6 , 5 2 0 , a n d 434 my ( F i g . 1 1 a ) . A t r o u g h a t 460 my i s a p p a r e n t , i n d i -c a t i n g t h e p r e s e n c e o f a f l a v o p r o t e i n i n t h i s f r a c t i o n . Sweat a n d Y o u n g (128) h a v e o b t a i n e d a p u r i f i e d f l a v o p r o t e i n f r o m a f r a c t i o n p r e c i p i t a t e d by 70% s a t u r a t i o n w i t h ammonium s u l f a t e , b u t f o u n d t h a t i t d i d n o t e n h a n c e l l g - h y d r o x y l a t i o n . Cammer a n d E s t a b r o o k (85): h a v e s u g g e s t e d t h a t t h e d e c r e a s e i n a b s o r b a n c e a t a b o u t 460 my i s p o s s i b l y d u e t o r e d u c t i o n o f nonheme i r o n p r o t e i n o f t h e 1 1 6 - h y d r o x y l a s e p a t h w a y as w e l l a s f l a v o p r o t e i n . K i m u r a a n d S u z u k i (59) h a v e d e m o n s t r a t e d t h a t t h e nonheme i r o n c o m p o n e n t i s p r e s e n t i n t h e f r a c t i o n p r e c i p i t a t i n g b e t w e e n '60% a n d 80% s a t u r a t i o n w i t h ammonium s u l f a t e . T h e r e f o r e b o t h f l a v o p r o t e i n a n d nonheme i r o n may b e c o n t r i b u t i n g t o t h e t r o u g h a t 460 my ( F i g . 1 1 a ) . T h e c a r b o n m o n o x i d e d i f f e r e n c e s p e c t r u m o f t h e f r a c t i o n p r e c i p i t a t i n g b e t w e e n 40% a n d 80% s a t u r a t i o n w i t h ammonium s u l f a t e i s c o m p l e x , h a v i n g a b s o r p t i o n maxima a t 6 0 8 , 5 7 0 , 5 3 3 , 4 2 2 , a n d 412 my a n d a p r o n o u n c e d minimum a t 436 my ( F i g . l i b ) . T h e s e a b s o r p t i o n maxima do n o t c o r r e s p o n d t o t h o s e o f t h e c a r b o n m o n o x i d e c o m p l e x o f a n y p r e v i o u s l y d e s c r i b e d c y t o c h r o m e a n d p e r h a p s r e p r e s e n t c a r b o n m o n o x i d e c o m p l e x e s o f c y t o c h r o m e d e g r a d a t i o n p r o d u c t s f o r m e d d u r i n g p r e p a r a t i o n o f t h e m i t o c h o n -d r i a l a c e t o n e p o w d e r o r t o d e n a t u r a t i o n o f h e m o p r o t e i n s s u c h as c y t o c h r o m e & t o a f o r m c a p a b l e o f b i n d i n g c a r b o n m o n o x i d e ( 9 0 ) . H e m o g l o b i n i s a l s o l i k e l y t o be p r e s e n t i n t h i s - 65 -ammonium s u l f a t e f r a c t i o n . Omura et al. (57) h a v e c a r r i e d o u t a n ammonium s u l f a t e f r a c t i o n a t i o n o f a n 113 - h y d r o x y l a s e p r e p a r e d f r o m s o n i c a t e d a d r e n a l m i t o c h o n d r i a . T h e y f o u n d t h a t t h e 60-80% ammonium s u l f a t e f r a c t i o n c o n t a i n e d nonheme i r o n p r o t e i n , f l a v o p r o t e i n , c y t o c h r o m e c, a n d a l a r g e amount o f h e m o g l o b i n . T h e c o m -p l e x s p e c t r a o f t h e 40-80% ammonium s u l f a t e f r a c t i o n ( F i g s . 11a a n d l i b ) o b t a i n e d i n t h e p r e s e n t s t u d i e s a l s o i n d i c a t e s t h e p r e s e n c e o f s e v e r a l c o m p o n e n t s i n t h i s f r a c t i o n . (d) 113 - H y d r o x y l a s e f r o m S o n i c a t e d A d r e n a l M i t o c h o n d r i a Enzyme s y s t e m s c a p a b l e o f i n t r o d u c i n g a h y d r o x y l f u n c t i o n i n t o t h e 113 p o s i t i o n o f t h e s t e r o i d n u c l e u s h a v e b e e n p r e -p a r e d b y v a r i o u s m e t h o d s ( 3 1 - 3 3 ) . Sharma et al. (33) h a v e r e p o r t e d t h a t t h e 113 - h y d r o x y l a s e e x t r a c t e d f r o m a n a c e t o n e p o w d e r o f b e e f a d r e n a l m i t o c h o n d r i a h a s a l o w e r a c t i v i t y t h a n t h e enzyme s y s t e m s o l u b i l i z e d by u l t r a s o n i c t r e a t m e n t o f a d r e n a l ' m i t o c h o n d r i a . T h e r e f o r e s t u d i e s w i t h a n 113 - h y d r o x y -l a s e p r e p a r e d b y t h e m e t h o d o f S h a r m a et al. w e r e u n d e r t a k e n i n o r d e r t o c o m p a r e t h e p r o p e r t i e s o f t h i s s y s t e m w i t h t h e a c e t o n e p o w d e r p r e p a r a t i o n . ( i ) NADPH o x i d a t i o n a n d 1 1 3 - h y d r o x y l a t i o n b y t h e s o n i c a t e d a d r e n a l m i t o c h o n d r i a p r e p a r a t i o n . NADPH i s o x i d i z e d by t h e enzyme p r e p a r e d f r o m s o n i c a t e d m i t o c h o n d r i a i n t h e a b s e n c e o f a d d e d s t e r o i d s u b s t r a t e ( F i g . 12) , t h e r a t e o f o x i d a t i o n b e i n g 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 . Upon a d d i t i o n o f e x c e s s q u a n t i t i e s o f - 66 -1.2 INCUBATION T IME ,m in F i g . 1 2 . NADPH o x i d a t i o n by a n enzyme p r e p a r a t i o n o b t a i n e d b y u l t r a c e n t r i f u g a t i o n o f s o n i c a t e d a d r e n a l m i t o c h o n d r i a . E a c h r e a c t i o n f l a s k c o n t a i n e d : 0 . 0 5 m l o f p r o p y l e n e g l y c o l ; 1 .0 m l o f T r i s M g C l 2 b u f f e r ; 2 . 0 m l o f a 0 . 1 5 4 M s o l u -t i o n o f K C 1 ; a n d 1 5 , 2 0 , o r 30 mg of . t h e l y o p h i l i z e d enzyme p r e p a r a t i o n . 0 . 9 y m o l e o f NADPH d i s s o l v e d i n 0 . 2 m l o f T r i s -M g C l 2 b u f f e r was a d d e d a t z e r o t i m e . A m o u n t o f enzyme p r e p a r a t i o n a d d e d (mg): 15 ( © ) ; 20 ( A ) ; 30 (o) ; 2 0 , p l u s 0 . 6 y m o l e o f d e o x y c o r t i c o s t e r o n e ( A ) . d e o x y c o r t i c o s t e r o n e (200 u g ) , no s i g n i f i c a n t i n c r e a s e i n t h e r a t e o f o x i d a t i o n o f NADPH i s o b s e r v e d ( F i g . 1 2 ) . Thus t h i s s y s t e m d i f f e r s i n t h i s r e s p e c t t o t h e a c e t o n e powder p r e p a r a -t i o n w h e r e a d d i t i o n o f s t e r o i d s u b s t r a t e p r o d u c e s a s u b s t a n -t i a l i n c r e a s e i n t h e r a t e o f NADPH o x i d a t i o n ( F i g . 4 ) . The s o n i c a t e d a d r e n a l m i t o c h o n d r i a l enzyme p r e p a r a t i o n d o e s c a t a -l y z e t h e 1 1 3-hydroxylation o f d e o x y c o r t i c o s t e r o n e . I n c u b a -t i o n o f t h e p r e p a r a t i o n (25 mg d i s s o l v e d i n 2.0 m l o f a 0.154 M s o l u t i o n o f K C l ) i n t h e p r e s e n c e o f 1.0 m l o f T r i s - M g C l 2 b u f f e r medium, 0.57 umole o f d e o x y c o r t i c o s t e r o n e , a n d 3 u m o l e s o f NADPH r e s u l t s i n t h e f o r m a t i o n o f 0.09 umole o f c o r t i c o -s t e r o n e a f t e r 30 m i n u t e s o f i n c u b a t i o n . I f t h e s t o i c h i o -m e t r y b e t w e e n 1 1 3-hydroxylation a n d NADPH o x i d a t i o n i s i n d e e d one m o l e o f h y d r o x y l a t e d s t e r o i d f o r m e d f o r e a c h m o l e o f NADPH o x i d i z e d , t h e n t h e t o t a l amount o f NADPH t h a t w o u l d be o x i d i z e d i n t h e 1 1 3-hydroxylation o f d e o x y c o r t i c o s t e r o n e t o c o r t i c o s t e r o n e w o u l d be 0.09 u m o l e . Thus o n l y a s l i g h t i n -c r e a s e i n t h e r a t e o f NADPH o x i d a t i o n w o u l d be a c h i e v e d d u r i n g t h e c o u r s e o f 1 1 3-hydroxylation. S u c h an i n c r e a s e may n o t be m e a s u r a b l e b y t h e e x p e r i m e n t a l m e t h o d s e m p l o y e d ( F i g . 1 2 ) . T h e s e r e s u l t s s u g g e s t t h a t t h e s u b s t a n t i a l i n -c r e a s e i n t h e r a t e o f NADPH o x i d a t i o n p r o d u c e d b y a d d i t i o n o f d e o x y c o r t i c o s t e r o n e t o t h e a c e t o n e powder p r e p a r a t i o n ( F i g . 4) i s n o t e n t i r e l y a r e s u l t o f 1 1 3 - h y d r o x y l a t i o n o f t h e s t e r o i d s u b s t r a t e . The p r e s e n c e o f c y t o c h r o m e P-450 i n t h e 1 1 3 - h y d r o x y l a s e p r e p a r e d b y t h e m e t h o d o f Sharma et- al. (33) h a s b e e n r e p o r t e d - 68 -( 5 6 , 5 7 , 8 6 ) . The c a r b o n m o n o x i d e d i f f e r e n c e s p e c t r u m o f t h i s enzyme s y s t e m p r e p a r e d f o r t h e p r e s e n t s t u d i e s was t h e r e f o r e d e t e r m i n e d . T h i s s p e c t r u m , i l l u s t r a t e d i n F i g u r e 1 3 , i s o b -t a i n e d a f t e r r e d u c t i o n o f t h e enzyme p r e p a r a t i o n w i t h NADPH. The i n t e n s e a b s o r p t i o n b a n d w i t h a maximum a t 450 my i s t h e d i s t i n c t f e a t u r e o f t h e s p e c t r u m a n d i n d i c a t e s t h e p r e s e n c e o f c y t o c h r o m e P-450. I n c o n t r a s t t o t h e a c e t o n e powder p r e -p a r a t i o n ( F i g s . 8 and 10b) t h e a b s o r p t i o n b a n d a t 420 my i s o n l y a m i n o r c o m ponent o f t h e c a r b o n m o n o x i d e d i f f e r e n c e s p e c t r u m o f t h e s o n i c a t e d enzyme p r e p a r a t i o n . The r e s u l t s o f t h e s e e x p e r i m e n t s ( F i g s . 12 a n d 13) i n d i c a t e t h a t t h e o x i d a t i o n o f NADPH i s a c c o m p a n i e d b y t h e r e d u c t i o n o f c y t o -chrome P-450, b e c a u s e o n l y t h e r e d u c e d f o r m o f t h i s hemo-p r o t e i n w i l l b i n d c a r b o n m o n o x i d e . C y t o c h r o m e P-450 i s e x t r e m e l y a u t o x i d i z a b l e ( 1 2 9 ) . The c y t o c h r o m e P-450 r e d u c e d d u r i n g t h e o x i d a t i o n o f NADPH w o u l d be r a p i d l y r e o x i d i z e d . T h e r e f o r e t h e s m a l l amounts o f c y t o c h r o m e P-450 p r e s e n t i n -5 t h e s o n i c a t e d enzyme p r e p a r a t i o n (5.4 x 10 ymole/mg o f powder) may f u n c t i o n c a t a l y t i c a l l y t o b r i n g a b o u t t h e e x t e n -s i v e o x i d a t i o n o f NADPH t h a t i s o b s e r v e d ( F i g . 1 2 ) . A c o m p a r i s o n o f t h e 1 1 3 - h y d r o x y l a s e a c t i v i t y a n d c y t o -c hrome P-450 c o n t e n t o f a c e t o n e powder p r e p a r a t i o n and t h e l y o p h i l i z e d s o n i c a t e p r e p a r a t i o n a r e shown i n T a b l e V. The l y o p h i l i z e d s o n i c a t e p r e p a r a t i o n h a s a p p r o x i m a t e l y t w i c e t h e 1 1 3 - h y d r o x y l a s e a c t i v i t y p e r m i l l i g r a m o f enzyme powder a s t h e a c e t o n e powder p r e p a r a t i o 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 e m p l o y e d . I n a d d i t i o n t h e s o n i c a t e d p r e p a r a t i o n c o n -t a i n s f i v e t i m e s t h e amount o f c y t o c h r o m e P-450 a s t h e - 69 -450 » » 1 1——i 1 1 i i i i 400 420 440 460 480 500 W A V E L E N G T H , m n F i g . 13. NADPH r e d u c e d c a r b o n m o n o x i d e d i f f e r e n c e s p e c t r u m o f a n e n z y m e p r e p a r a t i o n o b t a i n e d b y u l t r a c e n t r i f u g a t i o n o f s o n i c a t e d a d r e n a l m i t o c h o n d r i a . E a c h c u v e t t e c o n t a i n e d : 0.05 m l o f p r o p y l e n e g l y c o l ; 1.0 m l o f T r i s - M g C l 2 b u f f e r ; 1.95 m l o f a 0.154 M s o l u t i o n o f K C l ; 25 mg o f l y o p h i l i z e d e n z y m e p r e p a r a t i o n ; a n d NADPH (1.1 u m o l e s ) d i s s o l v e d i n 0.1 m l o f T r i s - M g C l 2 b u f f e r . A f t e r 5 m i n u t e s o f i n c u b a t i o n , c a r b o n m o n o x i d e w a s b u b b l e d t h r o u g h t h e s a m p l e c u v e t t e r e a c t i o n m i x t u r e f o r 60 s e c o n d s a n d t h e d i f f e r e n c e s p e c t r u m w a s r e c o r d e d . T A B L E V C o m p a r i s o n o f t h e 1 1 3 - h y d r o x y l a s e a c t i v i t y a n d c y t o c h r o m e P - 4 5 0 c o n t e n t i n t h e enzyme s y s t e m s p r e p a r e d b y e x t r a c t i o n o f a d r e n a l m i t o c h o n d r i a l a c e t o n e powder a n d b y u l t r a c e n t r i f u g a t i o n o f s o n i c a t e d a d r e n a l m i t o c h o n d r i a Enzyme p r e p a r a t i o n 1 1 3 - h y d r o x y l a s e a c t i v i t y — C y t o c h r o m e P - 4 5 0 c o n t e n t — P e r mg enzyme powder ( x l O 5 ) P e r mg p r o t e i n ( x l O 5 ) P e r mg enzyme p o w d e r ( x l O 5 ) P e r mg p r o t e i n ( x l O 5 ) M i t o c h o n d r i a l a c e t o n e p o w d e r 4 . 9~- 22 0 .98 4 . 4 L y o p h i l i z e d powder o f s o n i c a t e d m i t o c h o n d r i a 9 . 0 38 5 .4 23 — 1 1 3 - h y d r o x y l a s e a c t i v i t y = y m o l e c o r t i c o s t e r o n e f o r m e d / m i n / m g o f enzyme p o w d e r o r p r o t e i n . — c y t o c h r o m e P - 4 5 0 c o n t e n t = y m o l e / m g o f enzyme powder o r p r o t e i n . 1 1 3 - h y d r o x y l a s e a c t i v i t y was d e t e r m i n e d f r o m i n c u b a t i o n s c a r r i e d o u t a s d e s -c r i b e d i n F i g . 6. C y t o c h r o m e P - 4 5 0 c o n t e n t o f t h e m i t o c h o n d r i a l a c e t o n e powder p r e p a r a t i o n was e s t i m a t e d f r o m t h e d a t a shown i n F i g . 1 0 b . C y t o c h r o m e P - 4 50 c o n -t e n t o f t h e s o n i c a t e d m i t o c h o n d r i a l p r e p a r a t i o n was d e t e r m i n e d as shown i n F i g . 13 e x c e p t t h a t s o d i u m d i t h i o n i t e was e m p l o y e d i n s t e a d o f NADPH t o r e d u c e t h e hemo-p r o t e i n s . - 7 1 -a c e t o n e p o w d e r . S i m i l a r r e s u l t s a r e o b t a i n e d when l l g - h y d r o x y -l a s e a c t i v i t y a n d c y t o c h r o m e P - 4 5 0 c o n t e n t a r e e x p r e s s e d o n t h e b a s i s o f p r o t e i n c o n t e n t ( T a b l e V ) . C y t o c h r o m e P - 4 5 0 was e s t i m a t e d u s i n g t h e d a t a o f Omura a n d S a t o (89) who d e t e r -m i n e d a n e x t i n c t i o n c o e f f i c i e n t f o r c y t o c h r o m e P - 4 5 0 o f 91 cm ^ mM o n a p r o t o h e m e b a s i s , u t i l i z i n g t h e change- ' - in o p -t i c a l d e n s i t y b e t w e e n 450 a n d 490 my i n t h e c a r b o n m o n o x i d e d i f f e r e n c e s p e c t r u m . A l t h o u g h t h e r e i s a f i v e - f o l d d i f f e r e n c e i n t h e c y t o c h r o m e P - 4 5 0 c o n t e n t o f t h e enzyme s y s t e m s p r e p a r e d b y t h e two d i f f e r e n t m e t h o d s , t h e r e i s o n l y a t w o - f o l d d i f -f e r e n c e i n t h e o v e r a l l l l g - h y d r o x y l a s e a c t i v i t y . T h u s i t w o u l d seem t h a t t h e c y t o c h r o m e P - 4 5 0 c o n t e n t i s n o t t h e r a t e -l i m i t i n g f a c t o r i n t h e h y d r o x y l a t i o n r e a c t i o n . ( i i ) E f f e c t o f a c e t o n e o n t h e s o n i c a t e d enzyme p r e p a r a -t i o n . T h e r e h a v e b e e n v a r i o u s r e p o r t s o n t h e d e c o m p o s i t i o n o f c y t o c h r o m e P - 4 5 0 by t r e a t m e n t w i t h o r g a n i c s o l v e n t s ( 8 1 , 9 0 , 9 3 ) . T h e r e f o r e i t i s l i k e l y t h a t t h e r e l a t i v e l y l o w r e -c o v e r y o f t h i s c y t o c h r o m e f r o m t h e a c e t o n e p o w d e r o f a d r e n a l m i t o c h o n d r i a i s a d i r e c t r e s u l t o f t h e m e t h o d s o f p r e p a r a t i o n o f t h e a c e t o n e p o w d e r . T h e e f f e c t o f a c e t o n e o n t h e c y t o c h r o m e P - 4 5 0 c o n t e n t o f 1 1 3 - h y d r o x y l a s e o b t a i n e d f r o m s o n i c a t e d a d r e -n a l m i t o c h o n d r i a was e x a m i n e d . E x t r a c t i o n o f t h e l y o p h i l i z e d enzyme p r e p a r a t i o n w i t h 100% a c e t o n e ( - 2 0 ° ) p r o d u c e s no a l t e r a t i o n i n t h e c y t o c h r o m e P - 4 5 0 c o n t e n t ( F i g . 1 4 ) . H o w e v e r , when t h e p r e p a r a t i o n i s s u b j e c t e d t o t h e same t r e a t m e n t w i t h a c e t o n e - w a t e r (9:1) - 7 2 -4 5 0 4 0 0 4 2 0 4 4 0 4 6 0 4 8 0 5 0 0 WAVELENGTH , m/i. F i g . 14. E f f e c t o f e x t r a c t i o n w i t h a c e t o n e o r a c e t o n e - w a t e r ( 9 : 1 v / v ) o n t h e c y t o c h r o m e P-4 50 c o n t e n t o f t h e s o n i c a t e d a d r e n a l m i t o c h o n d r i a enzyme p r e p a r a t i o n . The l y o p h i l i z e d enzyme p r e p a r a t i o n (200 mg) was e x t r a c -t e d w i t h a c e t o n e (2 x 10 ml) o r a c e t o n e - w a t e r , 9:1 v / v (2 x 10 ml) and t h e a c e t o n e - i n s o l u b l e m a t e r i a l v/as d r i e d in vacuo. The c y t o c h r o m e P-450 c o n t e n t o f 25 mg o f t h e e x t r a c t e d p r e -p a r a t i o n s was d e t e r m i n e d as d e s c r i b e d i n F i g . 13. S o d i u m d i t h i o n i t e was u s e d i n s t e a d o f NADPH t o r e d u c e t h e h e m o p r o t e i n s . U n e x t r a c t e d enzyme p r e p a r a t i o n ( ) ; a c e t o n e e x t r a c t e d enzyme p r e p a r a t i o n ( ) ; a c e t o n e - w a t e r e x t r a c t e d enzyme p r e p a r a t i o n ( ) . - 73 -t h e r e i s a d e c r e a s e i n c y t o c h r o m e P - 4 5 0 c o n t e n t a n d a c o r r e s -p o n d i n g i n c r e a s e i n c y t o c h r o m e P - 4 20 . I n a d d i t i o n t h e a b -s o r p t i o n maximum o f c y t o c h r o m e P - 4 2 0 i s s h i f t e d s l i g h t l y t o 418 my. T h u s t h e a c e t o n e t r e a t m e n t c a u s e s some a l t e r a t i o n o f t h e heme e n v i r o n m e n t o f c y t o c h r o m e P - 4 2 0 a n d may e x p l a i n t h e f a c t t h a t t h e c a r b o n m o n o x i d e d i f f e r e n c e s p e c t r u m o f c y t o -c h r o m e P - 4 2 0 i s o l a t e d f r o m t h e a c e t o n e powder o f a d r e n a l m i t o c h o n d r i a ( F i g . 10b) h a s a b s o r p t i o n maxima a t s l i g h t l y s h o r t e r w a v e l e n g t h s t h a n t h o s e r e p o r t e d f o r r a b b i t l i v e r m i -c r o s o m a l c y t o c h r o m e P - 4 2 0 b y Omura a n d S a t o ( 8 8 , 8 9 ) . T h e c o n -v e r s i o n o f c y t o c h r o m e P - 4 5 0 t o c y t o c h r o m e P - 4 2 0 b y t r e a t m e n t w i t h a c e t o n e - w a t e r (9 :1) s u g g e s t s t h a t t h e p h o s p h o l i p i d m o i e t y o f c y t o c h r o m e P - 4 5 0 i s b e i n g r e m o v e d . A c e t o n e - w a t e r (9 :1) h a s b e e n shown t o r e m o v e a t l e a s t 85% o f t h e p h o s p h o -l i p i d ' f r o m m i t o c h o n d r i a ( 1 0 9 , 1 3 0 ) . T h u s t h e low c y t o c h r o m e P - 4 5 0 c o n t e n t a n d 1 1 3 - h y d r o x y l a s e a c t i v i t y o f t h e enzyme s y s t e m o b t a i n e d f r o m t h e m i t o c h o n d r i a l a c e t o n e p o w d e r may b e a t t r i b u t e d t o t h e p r e p a r a t i o n o f t h e a c e t o n e p o w d e r . (e) S t u d i e s o n C y t o c h r o m e P - 4 5 0 i n t h e A d r e n a l M i t o c h o n d r i a l  A c e t o n e Powder P r e p a r a t i o n C y t o c h r o m e P - 4 5 0 i s p a r t i c u l a t e i n n a t u r e a n d p o s s e s s e s u n i q u e , s p e c t r a l p r o p e r t i e s . I t i s e a s i l y c o n v e r t e d b y a v a r i e t y o f a g e n t s t o c y t o c h r o m e P - 4 2 0 ( 8 8 - 9 4 ) . I n a d d i t i o n , c y t o c h r o m e P - 4 2 0 h a s b e e n f o u n d b y Omura a n d S a t o ( 8 8 , 8 9 ) t o be e x t r e m e l y l a b i l e u n d e r a e r o b i c c o n d i t i o n s . S t u d i e s o n t h e s t a b i l i t y o f c y t o c h r o m e s P - 4 5 0 a n d P - 4 2 0 u n d e r o u r e x p e r i -- 74 -m e n t a l c o n d i t i o n s w e r e t h e r e f o r e u n d e r t a k e n . ( i ) E f f e c t o f i n c u b a t i o n t i m e a n d t e m p e r a t u r e o n c y t o -c h r o m e s P - 4 50 a n d P - 4 2 0 . T h e e f f e c t o f i n c u b a t i o n t e m p e r a t u r e on c y t o c h r o m e s P -450 a n d P - 4 2 0 i s shown i n F i g u r e 1 5 . E a c h r e a c t i o n m i x t u r e was i n c u b a t e d a t t h e i n d i c a t e d t e m p e r a t u r e f o r t e n m i n u t e s w i t h c o n t i n u o u s s h a k i n g . S o l i d ammonium s u l f a t e was t h e n a d d e d a n d t h e f r a c t i o n c o n t a i n i n g c y t o c h r o m e s P - 4 5 0 a n d P - 4 2 0 was i s o l a t e d . T h e d a t a i n F i g u r e 15 i n d i c a t e t h a t at t e m p e r a t u r e s b e l o w 3 1 ° C c y t o c h r o m e P - 4 5 0 i s r e l a t i v e l y s t a b l e , t h e amount r e c o v e r e d a f t e r i n c u b a t i o n a t 3 1 ° C b e i n g o n l y s l i g h t l y l e s s t h a n t h a t r e c o v e r e d a f t e r i n c u b a t i o n a t 3 ° C . A c o n t r o l i n -c u b a t i o n , t o w h i c h ammonium s u l f a t e was a d d e d p r i o r t o i n c u b a -t i o n a t 3 ° C a n d i n w h i c h t h e a e r o b i c s h a k i n g s t e p was o m i t t e d , was p e r f o r m e d . T h e amount o f c y t o c h r o m e P - 4 5 0 r e c o v e r e d f r o m t h i s i n c u b a t i o n i s a l m o s t i d e n t i c a l t o t h a t o f a. r e a c t i o n m i x -t u r e i n c u b a t e d a t t h i s t e m p e r a t u r e w i t h s h a k i n g f o r 8 m i n u t e s . A s t h e i n c u b a t i o n t e m p e r a t u r e i s i n c r e a s e d f r o m 3 7 ° C t o 4 5 ° C , t h e r e i s a s h a r p d e c r e a s e i n t h e amount o f c y t o c h r o m e P - 4 50 r e c o v e r e d , i n d i c a t i n g t h e e x t r e m e l . a b - i l i t y o f t h i s h e m o p r o t e i n a t e l e v a t e d t e m p e r a t u r e s . T h e e f f e c t o f i n c u b a t i o n t e m p e r a t u r e o n c y t o c h r o m e P - 4 2 0 i s a l s o shown i n F i g u r e 1 5 . T h e amount o f c y t o c h r o m e P - 4 2 0 r e c o v e r e d a l s o t e n d s t o d e c r e a s e w i t h i n c r e a s i n g i n c u b a t i o n t e m p e r a t u r e . C y t o c h r o m e P - 4 2 0 l e v e l s a r e d i f f i c u l t t o i n t e r p r e t - 75 -~ 0.06 INCUBATION TEMPERATURE ,°C F i g . 1 5 . E f f e c t o f i n c u b a t i o n t e m p e r a t u r e on t h e r e c o v e r y o f c y t o c h r o m e s P-450 and P-420 f r o m t h e a d r e n a l m i t o c h o n d r i a l a c e t o n e powder enzyme p r e p a r a t i o n . E a c h r e a c t i o n f l a s k c o n t a i n e d : 0.15 m l o f p r o p y l e n e g l y c o l ; 2.40 m l o f T r i s - M g C l 2 b u f f e r ; a n d 3.9 m l o f enzyme p r e p a r a t i o n . A f t e r 10 m i n u t e s o f i n c u b a t i o n , s o l i d ammonium s u l f a t e was a d d e d t o g i v e 20% s a t u r a t i o n and c y t o c h r o m e s P-450 and P-420 w e r e i s o l a t e d and m e a s u r e d as d e s c r i b e d i n t h e m e t h o d s . C y t o c h r o m e P-450 (O) ; c y t o c h r o m e P-420 (©) . - 76 -h o w e v e r , a s t h e amount m e a s u r e d 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 i s t h e n e t r e s u l t o f t h e r a t e o f f o r m a t i o n o f c y t o c h r o m e P - 4 20 f r o m c y t o c h r o m e P - 4 50 a n d t h e r a t e o f b r e a k d o w n o f c y t o c h r o m e P - 4 2 0 i t s e l f . T h a t c y t o c h r o m e P - 4 2 0 i s t e m p e r a t u r e -i l a b i l e i s e v i d e n t f r o m t h e d a t a o b t a i n e d a t 4 5 ° C . A t t h i s t e m p e r a t u r e t h e c y t o c h r o m e P - 4 5 0 l e v e l h a s d e c r e a s e d s h a r p l y . I f c y t o c h r o m e P - 4 2 0 i s s t a b l e a t t h i s t e m p e r a t u r e one w o u l d e x p e c t a n i n c r e a s e i n t h e c y t o c h r o m e P - 4 2 0 l e v e l r e s u l t i n g f r o m i t s f o r m a t i o n f r o m c y t o c h r o m e P - 4 5 0 . T h i s i s n o t o b -s e r v e d h o w e v e r , t h e c y t o c h r o m e P - 4 2 0 c o n t e n t i s a l s o d i m i n i s h e d a t t h i s t e m p e r a t u r e when c o m p a r e d t o t h e c y t o c h r o m e P - 4 2 0 c o n t e n t a t l o w e r i n c u b a t i o n t e m p e r a t u r e s . The c y t o c h r o m e P - 4 5 0 c o n t e n t o f a r e a c t i o n m i x t u r e i n -c u b a t e d a t 3 7 ° C as a f u n c t i o n o f i n c u b a t i o n t i m e i s shown i n F i g u r e 1 6 . T h e r e i s p r o g r e s s i v e d e c r e a s e i n t h e c y t o c h r o m e P - 4 5 0 c o n t e n t w i t h i n c r e a s i n g i n c u b a t i o n t i m e . T h e d e c r e a s e i n c y t o c h r o m e P - 4 5 0 i s a c c o m p a n i e d b y a c o r r e s p o n d i n g i n -c r e a s e i n c y t o c h r o m e P - 4 2 0 , i l l u s t r a t i n g t h e p r e c u r s o r - p r o d u c t r e l a t i o n s h i p o f t h e two c y t o c h r o m e s . S i m i l a r r e s u l t s o n c y t o c h r o m e P - 4 5 0 b r e a k d o w n i n r a b b i t l i v e r m i c r o s o m e s h a v e b e e n r e p o r t e d b y I c h i k a w a a n d Yamano ( 9 4 ) . T h e y m e a s u r e d t h e c y t o c h r o m e P - 4 5 0 a n d P - 4 2 0 c o n t e n t o f m i c r o s o m e s i n c u b a t e d a e r o b i c a l l y a t 1 6 ° C , pH 7 .0 o v e r a number o f d a y s a n d o b -s e r v e d t h a t t h e s p o n t a n e o u s d e c o m p o s i t i o n o f c y t o c h r o m e P -450 g a v e a f i r s t - o r d e r c u r v e u n d e r t h e s e c o n d i t i o n s . F u r t h e r -m o r e t h e b r e a k d o w n o f c y t o c h r o m e P - 4 50 was n o t a c c o m p a n i e d b y a c o r r e s p o n d i n g i n c r e a s e i n c y t o c h r o m e P - 4 2 0 i n d i c a t i n g t h a t - 77 -0.06 0 .04 -0 .02 > a E O o m o o < DC o O o-to 2-m E < Q < o in i o in 0 4 8 12 INCUBATION T IME , min F i g . 16. C o n v e r s i o n o f c y t o c h r o m e P-450 t o c y t o c h r o m e P-a s a f u n c t i o n o f i n c u b a t i o n t i m e . 420 I n c u b a t i o n s w e r e c a r r i e d o u t as d e s c r i b e d i n F i g . 15. S o l i d ammonium s u l f a t e t o g i v e 20% s a t u r a t i o n was a d d e d a f t e r 0, 4 , 8, o r 12 m i n u t e s o f i n c u b a t i o n a n d c y t o c h r o m e s P-450 and P-420 w e r e i s o l a t e d and m e a s u r e d a s d e s c r i b e d i n t h e m e t h o d s . C y t o c h r o m e P-450 ( O ) ; c y t o c h r o m e P-420 (©). - 78 -t h i s h e m o p r o t e i n was a l s o l a b i l e . T h e r a t e o f b r e a k d o w n o f c y t o c h r o m e P - 4 50 i s o l a t e d f r o m t h e m i t o c h o n d r i a l a c e t o n e p o w d e r ( F i g . 16) i s much more r a p i d t h a n t h e r a t e o f b r e a k -down o f c y t o c h r o m e P - 4 50 o f l i v e r m i c r o s o m e s o b s e r v e d b y I c h i k a w a a n d Y a m a n o . The g r e a t e r r a t e o f c y t o c h r o m e P - 4 5 0 d e c o m p o s i t i o n i n t h e enzyme s y s t e m f r o m m i t o c h o n d r i a l a c e -t o n e p o w d e r c o u l d be due t o t h e h i g h e r i n c u b a t i o n t e m p e r a -t u r e ( 3 7 ° C) u s e d i n t h e s e s t u d i e s . T h e e n v i r o n m e n t o f t h e c y t o c h r o m e P - 4 5 0 i n i t s p a r t i -c u l a t e s t a t e may i n f l u e n c e t h e s t a b i l i t y o f t h i s h e m o p r o t e i n . E x a m i n a t i o n o f t h e s t a b i l i t y o f c y t o c h r o m e P - 4 5 0 i n t h e enzyme s y s t e m p r e p a r e d by s o n i c a t i o n o f a d r e n a l m i t o c h o n d r i a i n d i c a t e s t h a t t h i s c y t o c h r o m e i s s t a b l e u n d e r t h e same e x p e r i m e n t a l c o n d i t i o n s t h a t r e s u l t i n a 64% l o s s o f t h e c y t o c h r o m e P - 4 5 0 o f t h e a c e t o n e powder p r e p a r a t i o n . T h e r e -f o r e p r e p a r a t i o n o f t h e H B - h y d r o x y l a s e b y a c e t o n e t r e a t m e n t o f b e e f a d r e n a l m i t o c h o n d r i a r e s u l t s i n a p a r t i c u l a t e c y t o -c h r o m e P - 4 5 0 t h a t i s much more u n s t a b l e t h a n t h a t o b t a i n e d b y u l t r a s o n i c f r a g m e n t a t i o n o f a d r e n a l m i t o c h o n d r i a . T h i s i n s t a b i l i t y may be a r e s u l t o f r e m o v a l o f much o f t h e m i t o -c h o n d r i a l p h o s p h o l i p i d b y a c e t o n e t r e a t m e n t . T h e i n f l u e n c e o f NADPH o n t h e d e c o m p o s i t i o n o f c y t o -c h r o m e P - 4 5 0 was e x a m i n e d a n d t h e r e s u l t s a r e shown i n T a b l e V I . T h e p r e s e n c e o f NADPH i n t h e r e a c t i o n m i x t u r e t h r o u g h o u t t h e i n c u b a t i o n p e r i o d d o e s n o t s i g n i f i c a n t l y a f f e c t t h e r a t e o f c o n v e r s i o n o f c y t o c h r o m e P - 4 5 0 t o c y t o c h r o m e P - 4 2 0 . N i s h i -b a y a s h i a n d S a t o (84) h a v e r e p o r t e d t h a t u n d e r a e r o b i c s t e a d y T A B L E V I R e c o v e r y o f c y t o c h r o m e s P - 4 5 0 a n d P - 4 2 0 f r o m t h e m i t o c h o n d r i a l a c e t o n e p o w d e r enzyme p r e p a r a t i o n i n c u b a t e d i n t h e p r e s e n c e o f a b s e n c e o f NADPH I n c u b a t i o n t i m e ( m i n u t e s ) A A b s o r b a n c e 4 5 0 - 500 my 4 2 0 - 500 my No NADPH P l u s NADPH No NADPH P l u s NADPH 0 0 . 026 0 . 0 2 8 0 . 0 2 1 0 . 0 2 1 4 0 . 017 0 . 0 2 0 0 . 0 3 3 0 . 0 3 0 8 0. 011 0 . 0 1 2 0 . 0 4 1 0 . 0 4 0 12 0. 009 0 . 0 1 0 0 . 0 5 0 0 . 0 4 6 E a c h r e a c t i o n f l a s k c o n t a i n e d : 0 . 1 5 m l o f p r o p y l e n e g l y -c o l ; 2 . 2 5 m l o f T r i s - M g C l 2 b u f f e r ; 3 . 9 m l o f enzyme p r e p a r a t i o n NADPH ( 3 . 3 y m o l e s ) d i s s o l v e d i n 0 . 1 5 m l o f T r i s - M g C l 2 b u f f e r when a p p r o p r i a t e . A f t e r 0 , 4 , 8, o r 12 m i n u t e s o f i n c u b a t i o n , s o l i d ammonium s u l f a t e was a d d e d t o g i v e 20% s a t u r a t i o n a n d c y t o c h r o m e s P - 4 5 0 a n d P - 4 2 0 w e r e i s o l a t e d a n d m e a s u r e d a s d e s c r i b e d i n t h e m e t h o d s . - 80 " s t a t e c o n d i t i o n s a p p r o x i m a t e l y 30% o f t h e c y t o c h r o m e P - 4 5 0 ~ o f l i v e r m i c r o s o m e s was r e d u c e d by NADPH. Cammer a n d E s t a -b r o o k (85) h a v e o b s e r v e d t h a t t h e c y t o c h r o m e P - 4 5 0 o f b e e f a d r e n a l m i t o c h o n d r i a i s a l s o l a r g e l y i n t h e o x i d i z e d f o r m d u r i n g t h e a e r o b i c s t e a d y s t a t e o f d e o x y c o r t i c o s t e r o n e h y -d o x y l a t i o n . T h e r e f o r e t h e l a c k o f any d e m o n s t r a b l e e f f e c t o f NADPH o n t h e s t a b i l i t y o f c y t o c h r o m e P - 4 5 0 ( T a b l e V I ) i s n o t a n u n e x p e c t e d f i n d i n g . C y t o c h r o m e P - 4 5 0 i s e s s e n -t i a l l y i n t h e same f o r m ( i . e . o x i d i z e d form) i n t h e p r e s e n c e o f NADPH a s i n i t s a b s e n c e . Omura a n d S a t o ( 8 8 , 8 9 ) h a v e r e p o r t e d t h a t c y t o c h r o m e P - 4 2 0 i s m o r e u n s t a b l e i n t h e r e -d u c e d f o r m t h a n i n t h e o x i d i z e d f o r m u n d e r a e r o b i c c o n d i -t i o n s . S i m i l a r l y I m a i a n d S a t o (90) h a v e o b s e r v e d t h a t t h e c y t o c h r o m e P - 4 5 0 o f l i v e r m i c r o s o m e s was more s u s c e p t i b l e t o d e c o m p o s i t i o n by t h e a c t i o n o f n e u t r a l s a l t s when i n i t s r e d u c e d s t a t e . T h e p r e s e n t r e s u l t s w i t h c y t o c h r o m e P - 4 5 0 i n -d i c a t e t h a t t h e o x i d i z e d f o r m o f c y t o c h r o m e P - 4 5 0 i s a l s o e x t r e m e l y u n s t a b l e ( F i g . 16) a n d t h a t NADPH u n d e r a e r o b i c s t e a d y s t a t e c o n d i t i o n s d o e s n o t s i g n i f i c a n t l y a l t e r t h i s l a b i l i t y ( T a b l e V I ) . ( i i ) E f f e c t o f s t e r o i d s u b s t r a t e s o n c y t o c h r o m e s P - 4 5 0 a n d P - 4 2 0 . T h e a b i l i t y o f s u b s t r a t e s t o s t a b i l i z e enzymes h a s b e e n w e l l d o c u m e n t e d ( 1 3 1 - 1 3 3 ) . T h e r e f o r e i t seemed o f i m p o r t a n c e t o e x a m i n e t h e e f f e c t s o f b o t h s u b s t r a t e s a n d p r o d u c t s o f t h e 1 1 3 - h y d r o x y l a s e o n c y t o c h r o m e P - 4 5 0 . - 81 -I n c u b a t i o n o f t h e enzyme p r e p a r a t i o n a t 3 7 ° C i n t h e p r e s e n c e o f s u b s t r a t e d e o x y c o r t i c o s t e r o n e r e s u l t s i n a n i n -c r e a s e d r e c o v e r y o f c y t o c h r o m e P - 4 5 0 ( F i g . 1 7 ) . T h i s i n c r e a s e i s p r o p o r t i o n a l t o t h e amount o f d e o x y c o r t i c o s t e r o n e p r e s e n t a n d i s m a x i m a l a t d e o x y c o r t i c o s t e r o n e c o n c e n t r a t i o n s i n t h e r e g i o n o f t h e f o r l l & - h y d r o x y l a t i o n ( 5 . 5 u m o l e s / 1 ) . T h e s e r e s u l t s s u g g e s t t h a t d e o x y c o r t i c o s t e r o n e i s b i n d i n g t o c y t o -c h r o m e P - 4 50 a n d p r o t e c t i n g i t f r o m d e g r a d a t i o n p e r h a p s b y s h i e l d i n g some l a b i l e g r o u p o r by p r o d u c i n g some c o n f o r m a -t i o n a l c h a n g e i n t h e c y t o c h r o m e . T h e r e i s now s u b s t a n t i a l s p e c t r o p h o t o m e t r i c e v i d e n c e t h a t s t e r o i d s u b s t r a t e s do b i n d t o c y t o c h r o m e P - 4 5 0 ( 9 5 - 9 8 ) . T h e p r o t e c t i v e e f f e c t o f d e o x y -c o r t i c o s t e r o n e o n c y t o c h r o m e P - 4 5 0 i l l u s t r a t e d i n F i g u r e 17 i s i n d i c a t i v e o f s t e r o i d b i n d i n g t o c y t o c h r o m e P - 4 5 0 . O t h e r s t e r o i d s w e r e a l s o e x a m i n e d f o r t h e i r e f f e c t o n c y t o c h r o m e P - 4 5 0 . C o r t i c o s t e r o n e , t h e p r o d u c t o f 1 1 6 - h y d r o x y -l a t i o n o f d e o x y c o r t i c o s t e r o n e , h a s no p r o t e c t i v e e f f e c t a t a l l c o n c e n t r a t i o n s t e s t e d ( F i g . 1 7 ) . A n d r o s t e n e d i o n e , w h i c h h a s b e e n shown by Sharma et al. (41) t o be a c o m p e t i t i v e i n -h i b i t o r o f 1 1 6 - h y d r o x y l a t i o n o f d e o x y c o r t i c o s t e r o n e , p r o t e c t s c y t o c h r o m e P - 4 5 0 i n a m a n n e r a n a l o g o u s t o t h a t o f d e o x y c o r t i -c o s t e r o n e ( F i g . 1 8 ) . T h e p r e s e n c e o f a n 1 1 - o x o g r o u p i n a d r e n o s t e r o n e c o m p l e t e l y e l i m i n a t e s t h i s p r o t e c t i v e e f f e c t ( F i g . 1 8 ) . T h e p r o t e c t i v e e f f e c t s o f a n d r o s t e n e d i o n e a n d d e o x y c o r t i c o s t e r o n e o n c y t o c h r o m e P - 4 5 0 , a n d t h e c o m p e t i t i v e i n h i b i t i o n o f 1 1 6 - h y d r o x y l a t i o n o f d e o x y c o r t i c o s t e r o n e b y a n d r o s t e n e d i o n e d e m o n s t r a t e d b y S h a r m a et al. (41) s u g g e s t - 82 -0.05 d E o o m I o m iLl o < m DC o in CD < 0.03 0.02 -0.01 -S T E R O I D C O N C E N T R A T I O N . n,M0LES/l F i g . 1 7 . R e c o v e r y o f c y t o c h r o m e P - 4 5 0 f r o m t h e m i t o c h o n d r i a l a c e t o n e p o w d e r enzyme p r e p a r a t i o n a f t e r i n c u b a t i o n i n t h e p r e s e n c e o f d e o x y c o r t i c o s t e r o n e o r c o r t i c o s t e r o n e . E a c h r e a c t i o n f l a s k c o n t a i n e d : s t e r o i d d i s s o l v e d i n 0 . 1 5 m l o f p r o p y l e n e g l y c o l t o g i v e t h e i n d i c a t e d f i n a l c o n -c e n t r a t i o n ; 2 . 1 m l o f T r i s - M g C l 2 b u f f e r ; 3 . 9 m l o f enzyme p r e p a r a t i o n ; a n d 0 . 1 5 m l o f a d i l u t e a l c o h o l i c KOH s o l u t i o n . E a c h r e a c t i o n f l a s k was p r e - i n c u b a t e d f o r 8 m i n u t e s . NADPH ( 3 . 3 p ino l e s ) d i s s o l v e d i n 0 . 1 5 m l o f T r i s - M g C l 2 b u f f e r was a d d e d . A f t e r 90 s e c o n d s o f i n c u b a t i o n , s o l i d ammonium s u l f a t e was a d d e d t o g i v e 20% s a t u r a t i o n a n d c y t o c h r o m e P - 4 5 0 t h e n was i s o l a t e d and m e a s u r e d as d e s c r i b e d i n t h e m e t h o d s . S t e r o i d a d d e d : d e o x y c o r t i c o s t e r o n e ( o ) ; c o r t i c o s t e r o n e (n) . - 83 -d E O o in 0.03 i r o m 0.02 LL) o 2! < CD 0.01 -or o in m 0 5 10 20 4 0 6 0 8 0 [STEROID] , j imoles/l F i g . 18. Recovery of cytochrome P-450 from the m i t o c h o n d r i a l acetone powder enzyme p r e p a r a t i o n a f t e r i n c u b a t i o n i n the presence of androstenedione or adrenosterone. Incubations were c a r r i e d out as des c r i b e d i n F i g . 17. S t e r o i d added: androstenedione (o); adrenosterone ( o ) . < 0 - 84 -t h a t t h e s e s t e r o i d s b i n d a t t h e same s i t e o n t h e c y t o c h r o m e P - 4 5 0 m o l e c u l e . I m a i a n d S a t o (90) h a v e f o u n d , d u r i n g s t u d i e s o n s u b s t r a t e i n t e r a c t i o n w i t h c y t o c h r o m e P - 4 5 0 i n r a b b i t l i v e r m i c r o s o m e s , t h a t h i g h c o n c e n t r a t i o n s o f t h e s u b s t r a t e a n i l i n e r e s u l t e d i n some c o n v e r s i o n o f c y t o c h r o m e P - 4 5 0 t o c y t o c h r o m e P - 4 2 0 . T h e e f f e c t o f h i g h s u b s t r a t e d e o x y c o r t i c o s t e r o n e c o n c e n t r a -t i o n s u n d e r o u r e x p e r i m e n t a l c o n d i t i o n s i s shown i n F i g u r e 1 9 . A t t h e h i g h e s t c o n c e n t r a t i o n t e s t e d , 400 y m o l e s / 1 , d e o x y -c o r t i c o s t e r o n e s t i l l p r o v i d e s t h e same d e g r e e o f p r o t e c t i o n o f c y t o c h r o m e P - 4 50 as a t l o w e r c o n c e n t r a t i o n s a n d t h e s u b -s t r a t e - p r o d u c e d d e c o m p o s i t i o n o f c y t o c h r o m e P - 4 5 0 n o t e d by I m a i a n d S a t o (90) i s n o t a p p a r e n t i n t h e d e o x y c o r t i c o s t e r o n e -c y t o c h r o m e P - 4 50 s y s t e m e m p l o y e d i n o u r e x p e r i m e n t s . I n an a t t e m p t t o e s t a b l i s h t h e m a n n e r i n w h i c h d e o x y -c o r t i c o s t e r o n e p r o t e c t s c y t o c h r o m e P - 4 5 0 , t h e r a t e s o f d e -c o m p o s i t i o n o f c y t o c h r o m e P - 4 5 0 i n t h e a b s e n c e a n d i n t h e p r e s e n c e o f d e o x y c o r t i c o s t e r o n e s u b s t r a t e w e r e c o m p a r e d ( F i g . 2 0 ) . I n t h e a b s e n c e o f d e o x y c o r t i c o s t e r o n e t h e r e i s a r a p i d i n i t i a l b r e a k d o w n o f c y t o c h r o m e P - 4 5 0 d u r i n g t h e f i r s t f o u r m i n u t e s o f i n c u b a t i o n . I n t h e p r e s e n c e o f d e o x y -c o r t i c o s t e r o n e t h i s r a t e o f d e c o m p o s i t i o n i s r e d u c e d b y a p -p r o x i m a t e l y o n e - h a l f . T h e r e f o r e t h e e l e v a t e d c y t o c h r o m e P -4 50 l e v e l s p r o d u c e d by d e o x y c o r t i c o s t e r o n e i n F i g u r e 17 a r e t h e r e s u l t o f a d i m i n u t i o n i n t h e r a t e o f d e c o m p o s i t i o n o f t h i s c y t o c h r o m e . T h e b i n d i n g o f d e o x y c o r t i c o s t e r o n e t o c y t o -c h r o m e P - 4 5 0 r e t a r d s t h e r a t e o f o x i d a t i v e d e c o m p o s i t i o n - 85 -~ 0 .06 < o in i o in <t 0 1 — 1 ' ' 1 ' ~ 0 2 0 0 4 0 0 [DEOXYCORTICOSTERONE] , / x m o l e s / l F i g . 1 9 . R e c o v e r y o f c y t o c h r o m e s P - 4 5 0 a n d P - 4 2 0 f r o m t h e m i t o c h o n d r i a l a c e t o n e p o w d e r enzyme p r e p a r a t i o n a f t e r i n c u b a -t i o n i n t h e p r e s e n c e o f h i g h c o n c e n t r a t i o n s o f d e o x y c o r t i c o s t e r -o n e . I n c u b a t i o n s 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 i n F i g . 1 7 . C y t o c h r o m e P - 4 5 0 ( o ) ; c y t o c h r o m e P - 4 2 0 ( © ) . - 86 -1 i i 1 1 0 4 8 12 INCUBATION TIME , min F i g . 20. E f f e c t o f d e o x y c o r t i c o s t e r o n e on t h e r a t e o f c y t o -c h r o m e P-450 d i s a p p e a r a n c e . E a c h r e a c t i o n f l a s k c o n t a i n e d : 0.15 m l o f p r o p y l e n e g l y c o l c o n t a i n i n g d e o x y c o r t i c o s t e r o n e (38.8 u m o l e s / 1 , f i n a l c o n c e n t r a t i o n ) o r 0.15 m l o f p r o p y l e n e g l y c o l ( c o n t r o l ) ; 2.25 m l o f T r i s - M g C l 2 b u f f e r ; 3.9 m l o f enzyme p r e p a r a t i o n ; a n d 0.15 m l o f a d i l u t e a l c o h o l i c KOH s o l u t i o n . A f t e r i n c u b a t i o n f o r 0, 4, 8, o r 12 m i n u t e s , s o l i d ammonium s u l f a t e was a d d e d t o e a c h f l a s k t o g i v e 20% s a t u r a t i o n and c y t o c h r o m e s P-450 an d P-420 w e r e i s o l a t e d and m e a s u r e d a s d e s c r i b e d i n t h e m e t h o d s . C y t o c h r o m e P-450: c o n t r o l (© ©) , p l u s d e o x y c o r t i c o -s t e r o n e (o o ) . C y t o c h r o m e P-420: c o n t r o l ( © - - - © ) , p l u s d e o x y c o r t i c o s t e r o n e ( o o ) . - 87 -o f t h i s h e m o p r o t e i n e i t h e r b y p r o t e c t i n g some l a b i l e g r o u p o f t h e c y t o c h r o m e , o r b y i n d u c i n g some c o n f o r m a t i o n a l c h a n g e w h i c h r e n d e r s t h e c y t o c h r o m e m o r e s t a b l e . M e a s u r e m e n t o f c y t o c h r o m e P - 4 2 0 l e v e l s i n t h e p r e s e n c e o f d e o x y c o r t i c o s t e r o n e ( 3 8 . 8 umoles/1) i n d i c a t e s , t h a t no s u b -s t r a t e p r o t e c t i o n i s a f f o r d e d t h i s h e m o p r o t e i n a t t h i s s u b -s t r a t e c o n c e n t r a t i o n ( F i g . 2 0 ) . I n t h e e x p e r i m e n t s w i t h e x t r e m e l y h i g h c o n c e n t r a t i o n s o f d e o x y c o r t i c o s t e r o n e (up t o 400 umoles/1) some p r o t e c t i o n o f c y t o c h r o m e P - 4 2 0 i s o b s e r v e d , t h e r e c o v e r y o f c y t o c h r o m e P - 4 2 0 b e i n g a b o u t 75% h i g h e r i n t h e p r e s e n c e o f d e o x y c o r t i c o s t e r o n e (400 umoles/1) t h a n i n i t s a b s e n c e ( F i g . 1 9 ) . T h i s p r o t e c t i o n o f c y t o c h r o m e P - 4 2 0 s u g g e s t s t h a t d e o x y c o r t i c o s t e r o n e c a n b i n d t o t h i s h e m o p r o t e i n b u t t h e s u b s t r a t e a f f i n i t y f o r c y t o c h r o m e P - 4 2 0 i s much l o w e r t h a n t h a t f o r c y t o c h r o m e P - 4 5 0 . I m a i a n d S a t o ( 9 0 , 9 8 ) h a v e o b s e r v e d t h e b i n d i n g o f t h e s u b s t r a t e a n i l i n e t o a p a r t i a l l y p u r i f i e d c y t o c h r o m e P - 4 2 0 f r o m r a b b i t l i v e r m i c r o s o m e s . T h e s u b s t r a t e c o n c e n t r a t i o n r e q u i r e d f o r h a l f - m a x i m a l b i n d i n g t o c y t o c h r o m e P - 4 2 0 was a p p r o x i m a t e l y 50 t i m e s h i g h e r t h a n t h a t o b s e r v e d f o r t h e b i n d i n g o f a n i l i n e t o c y t o c h r o m e P - 4 5 0 . T h e s e o b s e r v a t i o n s o f I m a i a n d S a t o c o n c u r w i t h t h o s e o f t h e p r e s e n t e x p e r i m e n t s . D e o x y c o r t i c o s t e r o n e a f f o r d s p r o t e c t i o n o f c y t o c h r o m e P - 4 2 0 o n l y a t c o n c e n t r a t i o n s much h i g h e r t h a n t h o s e w h i c h m a x i m a l l y s t a b i l i z e c y t o c h r o m e P - 4 5 0 . D I S C U S S I O N I n e a r l y s t u d i e s o n e n z y m a t i c 1 1 3 - h y d r o x y l a t i o n much - 88 -e f f o r t was expended by several groups of investigators on defining the cofactor requirements of the 118-hydroxylase. These studies indicated that the reaction was an aerobic one requiring, or stimulated by, one or more of NAD, NADPH, ATP, Mg + + as well as fumarate or other intermediates of the Krebs c i t r i c acid cycle (22-25). C l a r i f i c a t i o n of the func-t i o n of these i n d i v i d u a l components was provided indepen-dently by Grant (31) and by Sweat and Lipscomb (8) who demon-strated that the actual cofactor for llg-hydroxylation was NADPH and that the other compounds were i n fact concerned with the generation of NADPH. The absolute requirement of the llp-hydroxylase ex-tracted from a bovine adrenal mitochondrial acetone powder for NADPH has been demonstrated i n the present studies (Table I ) . Indeed s i g n i f i c a n t conversion of deoxycorticosterone to corticosterone required a high molar r a t i o of NADPH to steroid substrate. Spectrophotometric examination of NADPH oxidation by the enzyme preparation revealed that extensive oxidation of this cofactor occurred even i n the absence of steroi d sub-strat e . The oxidation of NADPH was found to be in s e n s i t i v e to potassium cyanide, Antimycin A, and amytal and was there-fore not proceeding v i a the c l a s s i c a l electron transport chain. In addition, spectrophotometric analysis of the enzyme preparation indicated that the only cytochrome of the c l a s s i c a l electron transport chain enzymatically reduced by NADPH was cytochrome c (Fig. 7). B e c a u s e t h e enzyme p r e p a r a t i o n e m p l o y e d i n t h e s e s t u d i e s i s a c r u d e e x t r a c t o f a m i t o c h o n d r i a l a c e t o n e p o w d e r , o x i -d a t i o n o f NADPH c a t a l y z e d b y enzymes n o t i n v o l v e d i n 1 1 6 -h y d r o x y l a t i o n i s a d i s t i n c t p o s s i b i l i t y . T h e N A D P H - r e d u c e d m i n u s o x i d i z e d d i f f e r e n c e s p e c t r u m o f t h e enzyme p r e p a r a t i o n ( F i g . 7) e x h i b i t e d a n a b s o r p t i o n s p e c t r u m s i m i l a r t o c y t o c h r o m e c . T h i s w o u l d i n d i c a t e t h e p r e s e n c e o f a n N A D P H - c y t o c h r o m e c r e d u c t a s e i n t h i s enzyme p r e p a r a t i o n w h i c h c o u l d i n p a r t a t l e a s t a c c o u n t f o r t h e o b s e r v e d o x i d a t i o n o f NADPH i n t h e a b s e n c e o f s t e r o i d s u b s t r a t e . C y t o c h r o m e P - 4 2 0 was a l s o shown t o be a c o m p o n e n t o f t h i s enzyme p r e p a r a t i o n . T h i s c y t o c h r o m e i s n o t a n o r m a l c o n s t i t u e n t o f a d r e n a l m i t o c h o n d r i a b u t i s p r o d u c e d b y t h e b r e a k d o w n o f c y t o c h r o m e P - 4 5 0 . T h i s d e c o m -p o s i t i o n c a n be b r o u g h t a b o u t b y d e t e r g e n t s , ( 8 8 , 8 9 ) , p h o s p h o -l i p a s e ( 8 8 , 8 9 ) , a n d o r g a n i c s o l v e n t s ( 8 1 , 9 0 , 9 3 ) a s w e l l a s o t h e r a g e n t s . I n t h e p r e s e n t s t u d i e s i t was f o u n d t h a t a c e t o n e -w a t e r (9 :1) c o u l d c a u s e t h e c o n v e r s i o n o f c y t o c h r o m e P - 4 5 0 t o c y t o c h r o m e P - 4 2 0 ( F i g . 14) a n d t h e c y t o c h r o m e P - 4 2 0 p r e s e n t i n t h e enzyme p r e p a r a t i o n was p r o d u c e d d u r i n g t h e f o r m a t i o n o f t h e m i t o c h o n d r i a l a c e t o n e p o w d e r . T h e b r e a k d o w n o f c y t o -c h r o m e P - 4 5 0 by a c e t o n e - w a t e r ( 9 : 1 ) , as w e l l as by d e t e r g e n t s a n d p h o s p h o l i p a s e i s a c c o m p a n i e d by t h e r e m o v a l o f p h o s p h o -l i p i d . C y t o c h r o m e P - 4 2 0 c a n be r e d u c e d e n z y m a t i c a l l y b y NADPH ( 5 9 , 6 0 , 8 8 ) b u t d o e s n o t f u n c t i o n i n 1 1 6 - h y d r o x y l a t i o n . T h e p r e s e n c e o f t h i s c y t o c h r o m e i n t h e enzyme s y s t e m e x t r a c t e d f r o m t h e a c e t o n e p o w d e r o f b o v i n e a d r e n a l m i t o c h o n d r i a c o u l d be r e s p o n s i b l e f o r much o f t h e o x i d a t i o n o f N A D P H , n e c e s s i t a t i n g - 90 -t h e a d d i t i o n o f e x c e s s i v e amounts o f t h i s c o f a c t o r t o demon-s t r a t e q u a n t i t a t i v e 1 1 3 - h y d r o x y l a t i o n o f a d d e d s t e r o i d s u b -s t r a t e . N A D P H - s u p p o r t e d 1 1 3 - h y d r o x y l a t i o n i s i n s e n s i t i v e t o t h e e l e c t r o n t r a n s p o r t i n h i b i t o r s a n t i m y c i n A and a m y t a l b u t i s i n h i b i t e d b y d i c u m a r o l ( T a b l e I V ) . T h i s i n h i b i t o r i s known t o u n c o u p l e o x i d a t i v e p h o s p h o r y l a t i o n i n t h e c l a s s i c a l e l e c -t r o n t r a n s p o r t c h a i n (122-124). However i t s a b i l i t y t o i n -h i b i t 1 1 3 - h y d r o x y l a t i o 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 e m p l o y e d i n t h e s e s t u d i e s i s u n u s u a l , f o r i t h a s b e e n demon-s t r a t e d t h a t t h e c l a s s i c a l e l e c t r o n t r a n s p o r t c h a i n i s n o t i n v o l v e d i n N A D P H - s u p p o r t e d 1 1 3 - h y d r o x y l a t i o n . I n h i b i t i o n o f 1 1 3 - h y d r o x y l a t i o n s u p p o r t e d by t h e K r e b s c y c l e i n t e r m e d i a t e , s u c c i n a t e , b y e l e c t r o n t r a n s p o r t i n h i b i t o r s a n d u n c o u p l i n g a g e n t s h a s b e e n o b s e r v e d (24,39,115,117,126,134). W i t h i n t h e a d r e n a l m i t o c h o n d r i o n a n e n e r g y - l i n k e d r e d u c t i o n o f NAD by s u c c i n a t e c a n o c c u r . A p y r i d i n e n u c l e o t i d e t r a n s h y d r o g e n a s e c a n t h e n b r i n g a b o u t t h e r e d u c t i o n o f NADP. T h a t t h e s e two r e a c t i o n s a r e e n e r g y - l i n k e d e x p l a i n s t h e s e n s i t i v i t y o f s u c c i n a t e - s u p p o r t e d 1 1 3 - h y d r o x y l a t i o n t o e l e c t r o n t r a n s p o r t i n h i b i t o r s a n d u n c o u p l i n g a g e n t s . I t i s b e l i e v e d t h a t i n a d r e n o c o r t i c a l m i t o c h o n d r i a t h e r e i s an i n t e r a c t i o n b e t w e e n t h e c l a s s i c a l r e s p i r a t o r y c h a i n a n d t h e 1 1 3 - h y d r o x y l a s e s y s t e m (39,115) w i t h t h e r e s u l t t h a t o x i d a t i o n o f s u b s t r a t e s by t h e c o n v e n t i o n a l r e s p i r a t o r y c h a i n c a n u l t i m a t e l y l e a d t o t r a n s f e r o f e l e c t r o n s t o t h e 1 1 3 - h y d r o x y l a s e p a t h w a y . H o w e v e r , N A D P H - s u p p o r t e d 1 1 3 - h y d r o x y l a t i o n i s n o t e n e r g y - l i n k e d . - 91 -T h e r e f o r e d i c u m a r o l m u s t be i n h i b i t i n g t h i s r e a c t i o n by a m e c h a n i s m d i s t i n c t f r o m i t s e f f e c t o n o x i d a t i v e p h o s p h o r y l a t i o n . A d e t a i l e d s t u d y on t h e m e c h a n i s m o f t h i s i n h i b i t i o n w i l l be d i s c u s s e d i n a l a t e r p a r t o f t h i s t h e s i s . C y t o c h r o m e P - 4 5 0 s e r v e s a s t h e t e r m i n a l o x i d a s e f o r t h e 1 1 6 - h y d r o x y l a t i o n o f d e o x y c o r t i c o s t e r o n e ( 5 6 , 5 7 , 7 2 ) a n d i t s p r e s e n c e i n a d r e n a l m i t o c h o n d r i a h a s b e e n w e l l e s t a b l i s h e d ( 5 7 , 6 9 - 7 1 ) . T h e 113 - h y d r o x y l a s e p r e p a r e d b y e x t r a c t i o n o f a n a d r e n a l m i t o c h o n d r i a l a c e t o n e p o w d e r w i t h a p o t a s s i u m c h l o r i d e s o l u t i o n c o n t a i n s t h i s c y t o c h r o m e ( F i g . 10b) a l -t h o u g h i n l e s s e r a m o u n t s t h a n t h e 113 - h y d r o x y l a s e p r e p a r e d by d i f f e r e n t m e t h o d s ( T a b l e V ) . T h i s h e m o p r o t e i n c a n o n l y be d e t e c t e d a f t e r f r a c t i o n a t i o n o f t h e enzyme p r e p a r a t i o n w i t h ammonium s u l f a t e . T h e c a r b o n m o n o x i d e d i f f e r e n c e s p e c -t r u m o f t h e f r a c t i o n c o n t a i n i n g c y t o c h r o m e P - 4 5 0 a l s o e x -h i b i t s a n a b s o r p t i o n maximum a t 420 my w h i c h may be due t o c o n t r i b u t i o n s b y c y t o c h r o m e P - 4 2 0 a n d h e m o g l o b i n . T h e c y t o c h r o m e P - 4 5 0 i s o l a t e d f r o m t h e a c e t o n e p o w d e r o f a d r e n a l m i t o c h o n d r i a i s e x t r e m e l y l a b i l e a n d u n d e r g o e s s p o n t a n e o u s d e c o m p o s i t i o n a t t e m p e r a t u r e s h i g h e r t h a n 3 0 ° C t o c y t o c h r o m e P - 4 2 0 ( F i g s . 15 a n d 1 6 ) . A s i m i l a r b r e a k -down o f c y t o c h r o m e P - 4 5 0 u n d e r a e r o b i c c o n d i t i o n s h a s b e e n o b s e r v e d by Omura a n d S a t o ( 8 9 ) , I c h i i et at. ( 8 0 ) , a n d I c h i k a w a a n d Yamano ( 9 4 ) . C y t o c h r o m e P - 4 2 0 i s a l s o u n s t a b l e a n d i s d e g r a d e d d u r i n g i n c u b a t i o n . T h e l o s s o f c y t o c h r o m e P - 4 2 0 i s a c c o m p a n i e d b y a l o s s o f t o t a l p r o t o h e m e ( 8 9 ) . Omura - 92 -a n d S a t o (89) o b s e r v e d t h a t b o t h c y t o c h r o m e s P - 4 50 a n d P - 4 2 0 w e r e s t a b i l i z e d u n d e r a n a e r o b i c c o n d i t i o n s s u g g e s t i n g t h a t t h e b r e a k d o w n o f t h e c y t o c h r o m e s i n v o l v e d i n t e r a c t i o n w i t h o x y g e n . I c h i k a w a a n d : Y a m a n o (94) a n d I c h i i et a l . (80) d e m o n -s t r a t e d t h a t t h e a e r o b i c d e c o m p o s i t i o n o f c y t o c h r o m e P - 4 5 0 was p r e v e n t e d i n t h e p r e s e n c e o f g l y c e r o l . R e s u l t s o f t h e p r e s e n t e x p e r i m e n t s i n d i c a t e t h a t c y t o c h r o m e P - 4 50 i s s t a b i -l i z e d i n t h e p r e s e n c e o f s t e r o i d s u b s t r a t e ( F i g s . 1 7 - 2 0 ) . T h i s e f f e c t a p p e a r s t o be s p e c i f i c f o r t h e s u b s t r a t e s o f t h e s t e r o i d 1 1 3 - h y d r o x y l a s e . The p r e s e n c e o f a h y d r o x y l o r oxo f u n c t i o n a t t h e C - l l p o s i t i o n o f t h e s t e r o i d n u c l e u s e l i m i n a t e s t h e p r o t e c t i v e e f f e c t . T h e p r e s e n c e o f a C - 1 7 s i d e c h a i n d o e s n o t a p p e a r i m p o r t a n t , h o w e v e r , a s a n d r o s t e n e d i o n e i s a s e f f e c t i v e a s d e o x y c o r t i c o s t e r o n e i n p r o t e c t i n g c y t o c h r o m e P - 4 5 0 . V e r y l i t t l e i s known a b o u t t h e s u b s t r a t e s p e c i f i c i t y o f c y t o c h r o m e P - 4 5 0 . M o r e e x t e n s i v e i n v e s t i g a t i o n s o f t h e t y p e o u t l i n e d i n F i g u r e s 17 a n d 18 c o u l d a i d i n t h e a s s i g n -ment o f t h e r e q u i s i t e s t r u c t u r a l a s p e c t s o f t h e s t e r o i d m o l e -c u l e i n v o l v e d i n enzyme b i n d i n g a n d s p e c i f i c i t y o f t h e 1 1 3 -h y d r o x y l a s e . T h e p r o t e c t i v e e f f e c t o f d e o x y c o r t i c o s t e r o n e o n c y t o c h r o m e P - 4 5 0 c o u l d p o s s i b l y a c c o u n t f o r t h e s t o i c h i o m e t r y o f g r e a t e r t h a n u n i t y o b s e r v e d f o r NADPH o x i d a t i o n a n d 1 1 3 - h y d r o x y l a t i o n ( F i g . 4 ) . I n c u b a t i o n s c o n t a i n i n g d e o x y c o r t i c o s t e r o n e w o u l d a l s o c o n t a i n more c y t o c h r o m e P - 4 50 t h a n t h o s e w i t h o u t d e o x y -c o r t i c o s t e r o n e . T h i s h i g h e r c y t o c h r o m e P - 4 50 c o n t e n t c o u l d - 93 -r e s u l t i n a g r e a t e r r a t e o f NADPH o x i d a t i o n e v e n t h o u g h i t may n o t be a s s o c i a t e d w i t h 1 1 6 - h y d r o x y l a t i o n . T h e m e c h a n i s m w h e r e b y d e o x y c o r t i c o s t e r o n e p r o t e c t s c y t o -c h r o m e P - 4 50 m u s t a w a i t f u r t h e r s t u d i e s o n t h e n a t u r e o f t h i s h e m o p r o t e i n . T h e c o n v e r s i o n o f c y t o c h r o m e P - 4 5 0 t o c y t o -c h r o m e P - 4 2 0 r e s u l t s f r o m a n a l t e r a t i o n o f t h e h y d r o p h o b i c n a t u r e o f t h i s h e m o p r o t e i n . T h e u n u s u a l s p e c t r a l p r o p e r t i e s o f c y t o c h r o m e P - 4 5 0 a r e b e l i e v e d t o be a r e s u l t o f h y d r o p h o b i c i n t e r a c t i o n o f t h e heme w i t h h y d r o p h o b i c a r e a s o f t h e P - 4 50 p r o t e i n o r w i t h l i p i d s p r e s e n t i n t h e c y t o c h r o m e P - 4 50 c o m p l e x . A n y a l t e r a t i o n o f t h i s i n t e r a c t i o n t h e r e f o r e r e s u l t s i n t h e c o n v e r s i o n o f c y t o c h r o m e P - 4 5 0 t o c y t o c h r o m e P - 4 2 0 . A s t h e s t e r o i d s u b s t r a t e s f o r 1 1 6 - h y d r o x y l a t i o n a r e h y d r o p h o b i c i n n a t u r e , i t i s r e a s o n a b l e t o assume t h a t t h e s e m o l e c u l e s c a n p e n e t r a t e t o t h e r e a c t i v e a r e a o f t h e c y t o c h r o m e P - 4 5 0 c o m -p l e x , i . e . , t h e a c t i v e s i t e , a n d m a i n t a i n t h e h y d r o p h o b i c n a t u r e o f t h i s c o m p l e x e i t h e r b y p r o t e c t i n g some l a b i l e g r o u p , o r by a l t e r i n g t h e c y t o c h r o m e t o a more s t a b l e c o n f o r m a t i o n . A s t h i s p r o t e c t i v e e f f e c t i s a f f o r d e d o n l y b y t h e s u b s t r a t e s t e r o i d s , a n d n o t b y t h e p r o d u c t s o f 1 1 6 - h y d r o x y l a t i o n ( e . g . , c o r t i c o s t e r o n e ) , t h e s u b s t r a t e b i n d i n g s i t e w o u l d a p p e a r t o be i n v o l v e d . T h e i n a b i l i t y o f c o r t i c o s t e r o n e a n d a d r e n o -s t e r o n e t o s t a b i l i z e c y t o c h r o m e P - 4 5 0 s u g g e s t s t h a t t h e s t a b i -l i z a t i o n i s n o t a g e n e r a l e f f e c t p r o d u c e d by a n y h y d r o p h o b i c c o m p o u n d . A n a l t e r n a t i v e m e c h a n i s m f o r i n c r e a s e d l e v e l s o f c y t o -c h r o m e P - 4 5 0 i n t h e p r e s e n c e o f s t e r o i d s u b s t r a t e s c o u l d be - 94 -t h e r e c o n v e r s i o n o f c y t o c h r o m e P - 4 2 0 t o c y t o c h r o m e P - 4 5 0 i n d u c e d by t h e a d d i t i o n o f s t e r o i d . I c h i k a w a a n d Yamano (94) h a v e o b s e r v e d t h e r e c o n v e r s i o n o f d e t e r g e n t a n d s u l f h y d r y l r e a g e n t - p r o d u c e d c y t o c h r o m e P - 4 2 0 t o c y t o c h r o m e P - 4 5 0 b y p o l y o l s s u c h as g l y c e r o l a n d e t h y l e n e g l y c o l , a n d b y g l u t a t h i o n e . T h e y d e m o n s t r a t e d r e c o n v e r s i o n a t t e m p e r a t u r e s as l o w as 0 ° C . A s i m i l a r d e o x y c o r t i c o s t e r o n e - i n d u c e d c o n v e r s i o n o f c y t o -c h r o m e P - 4 2 0 t o P - 4 5 0 w o u l d e x p l a i n t h e h i g h e r c y t o c h r o m e P - 4 5 0 l e v e l s o b t a i n e d i n t h e p r e s e n c e o f d e o x y c o r t i c o s t e r o n e i n t h e z e r o t i m e c o n t r o l i n c u b a t i o n ( m a i n t a i n e d a t 4 ° C) o f F i g u r e 2 0 , e v e n t h o u g h t h e h e m o p r o t e i n i s r e l a t i v e l y s t a b l e a t t h i s t e m p e r a t u r e ( F i g . 1 5 ) . T h e e x i s t e n c e o f c y t o c h r o m e P - 4 2 0 i n v a r i o u s f o r m s h a s b e e n s u g g e s t e d ( 8 1 ) . I t i s p o s s i b l e t h a t t h e b i n d i n g o f d e o x y c o r t i c o s t e r o n e t o one o f t h e c y t o -c h r o m e P - 4 2 0 s t a t e s c a n p r o d u c e a c o n f o r m a t i o n a l c h a n g e r e -s u l t i n g i n a r e f o r m a t i o n o f c y t o c h r o m e P - 4 5 0 p e r h a p s d u e t o a n i n c r e a s e i n h y d r o p h o b i c i n t e r a c t i o n s i n t h e a l t e r e d c y t o -c h r o m e P - 4 2 0 c o m p l e x . B o t h o f t h e a b o v e p o s t u l a t e d m e c h a n i s m s may b e c o n t r i b u t i n g t o t h e i n c r e a s e d l e v e l s o f c y t o c h r o m e P - 4 5 0 e v i d e n t i n t h e p r e s e n c e o f s t e r o i d s u b s t r a t e . - 95 -PART I I THE ROLE OF QUINONES IN 11g-HYDROXYLATION The e v i d e n c e p r e s e n t e d i n P a r t I o f t h i s t h e s i s s u g g e s t e d t h a t t h e i n h i b i t i o n o f 1 1 3 - h y d r o x y l a t i o n by d i c u m a r o l d i d n o t r e s u l t f r o m t h e u n c o u p l i n g o f o x i d a t i v e p h o s p h o r y l a t i o n by t h i s i n h i b i t o r . Numerous r e p o r t s h a v e b e e n p u b l i s h e d d e s c r i b -i n g d i c u m a r o l - s e n s i t i v e s y s t e m s i n a n i m a l t i s s u e s w h i c h c a n u t i l i z e q u i n o n e s as e l e c t r o n a c c e p t o r s and i n w h i c h q u i n o n e s c a n m e d i a t e t h e a e r o b i c o x i d a t i o n o f NADPH o r NADH by m o l e c u -l a r o x y g e n ( 6 2 , 1 0 9 , 1 3 5 - 1 4 1 ) . The q u i n o n e , Coenzyme Ch Q , i s known t o be p r e s e n t i n mammalian m i t o c h o n d r i a ( 1 4 2 ) . I t t h e r e -f o r e seemed p o s s i b l e t h a t t h e d i c u m a r o l i n h i b i t i o n o f l l g -h y d r o x y l a t i o n d e m o n s t r a t e d i n o u r e x p e r i m e n t s c o u l d be e x -p l a i n e d by i n h i b i t i o n o f some r e a c t i o n i n v o l v i n g a q u i n o n e ( v i t a m i n K o r coenzyme Q) compound. T h i s w o u l d i m p l y a f u n c -t i o n f o r v i t a m i n K o r coenzyme Q i n 1 1 3 - h y d r o x y l a t i o n . F i t c h and F o l k e r s (143) h a v e r e p o r t e d t h a t coenzyme Ch a h a s t h e c a p a c i t y t o s t a b i l i z e b i o l o g i c a l membranes. Thus t h e r e i s a p o s s i b l i t y t h a t coenzyme Ch 0 i s a s s o c i a t e d w i t h c y t o c h r o m e P-4 50 w h i c h i s a l s o membrane-bound. A l t e r n a t i v e l y a q u i n o n e may be f u n c t i o n i n g i n t h e e l e c t r o n t r a n s p o r t s e q u e n c e o f t h e 1 1 3 - h y d r o x y l a s e p a t h w a y . A s t u d y o f t h e p o s s i b l e i n v o l v e m e n t o f a v i t a m i n K o r c o enzyme Q - t y p e compound i n I I 3 - h y d r o x y l a t i o n was t h e r e f o r e u n d e r t a k e n . RESULTS (a) E f f e c t o f A c e t o n e - E x t r a c t e d L i p i d a n d M e n a d i o n e ( V i t a m i n K 3 ) o n NADPH O x i d a t i o n - 96 -D u r i n g t h e p r e p a r a t i o n o f t h e a d r e n a l m i t o c h o n d r i a l a c e -t o n e p o w d e r , a m a j o r i t y o f t h e p h o s p h o l i p i d s a n d q u i n o n e s a r e e x t r a c t e d f r o m t h e m i t o c h o n d r i a b y a c e t o n e ( 1 0 9 , 1 3 0 , 1 4 4 , 1 4 5 ) . I f t h e s e compounds a r e i n v o l v e d i n l l g - h y d r o x y l a t i o n a n d NADPH o x i d a t i o n t h e n t h e l l g - h y d r o x y l a s e p r e p a r e d f r o m t h e a c e t o n e powder w o u l d h a v e a r e d u c e d a c t i v i t y c o m p a r e d t o a s y s t e m p r e p a r e d by a n a l t e r n a t e m e t h o d ( T a b l e V ) . The t o t a l a c e t o n e - e x t r a c t e d f r a c t i o n was t h e r e f o r e e x a m i n e d f o r i t s e f f e c t on NADPH o x i d a t i o n . A d d i t i o n o f a s o n i c a t e d s u s p e n s i o n o f t h e a c e t o n e - e x t r a c t e d m a t e r i a l r e s u l t s i n a s u b s t a n t i a l i n c r e a s e i n t h e r a t e o f NADPH o x i d a t i o n ( F i g . 2 1 ) . T h i s i n c r e a s e d o x i d a t i o n o f NADPH i s d e p e n d e n t on t h e p r e s e n c e o f t h e enzyme p r e p a r a t i o n a s i s e v i -d e n c e d b y t h e l a c k o f NADPH o x i d a t i o n i n i t s a b s e n c e . A c e t o n e e x t r a c t s f r o m d i f f e r e n t a d r e n a l m i t o c h o n d r i a l a c e t o n e powder p r e p a r a t i o n s w e r e e x a m i n e d , a n d a l t h o u g h t h e a b s o l u t e s t i m u -l a t i o n o f NADPH o x i d a t i o n v a r i e d f r o m e x t r a c t t o e x t r a c t , s i m i l a r r e s u l t s t o t h o s e shown i n F i g u r e 21 w e r e g e n e r a l l y o b s e r v e d . K a m i n et al. (62) h a v e r e p o r t e d t h a t t h e NADPH o x i d a s e a c t i v i t y o f a p u r i f i e d N A DPH-cytochrome a r e d u c t a s e f r o m l i v e r m i c r o s o m e s c o u l d be m a r k e d l y s t i m u l a t e d b y l i p i d e x t r a c t s d e r i v e d f r o m l i v e r a n d a d r e n a l c o r t e x a l t h o u g h t h e y f o u n d t h e s t i m u l a t o r y e f f e c t t o be e x t r e m e l y v a r i a b l e . The p u r i f i e d enzyme a l s o e x h i b i t e d NADPH-menadione r e d u c t a s e a c -t i v i t y . NADPH o x i d a t i o n by t h e enzyme p r e p a r a t i o n e m p l o y e d i n t h e p r e s e n t s t u d i e s i s a l s o s t i m u l a t e d b y m e n a d i o n e ( 2 -m e t h y l - 1 , 4 - n a p h t h o q u i n o n e ) ( F i g . 2 2 ) . Thus NADPH o x i d a t i o n - 97 -1.4 INCUBATION T IME , min F i g . 2 1 . E f f e c t o f t h e a c e t o n e - e x t r a c t e d m i t o c h o n d r i a l l i p i d f r a c t i o n on NADPH o x i d a t i o n . E a c h r e a c t i o n f l a s k c o n t a i n e d : 1.0 ml o f T r i s ~ M g C l 2 b u f f e r ; 2 m l o f enzyme p r e p a r a t i o n ; 0.02 m l o f a s o n i c a t e d l i p i d s u s -p e n s i o n c o n t a i n i n g 150 mg l i p i d / m l ( t h e c o n t r o l r e a c t i o n f l a s k r e c e i v e d 0.02 m l o f T r i s - M g C l 2 b u f f e r ) . 0.9 umole o f NADPH i n 0.1 m l o f T r i s - M g C l 2 b u f f e r was a d d e d t o e a c h r e a c t i o n f l a s k a t z e r o t i m e . C o n t r o l (©); p l u s l i p i d , 0.96 mg/ml, ( o ) ; l i p i d c o n t r o l , 2 m l o f a 0.154 M K C l s o l u t i o n i n s t e a d o f enzyme p r e p a r a t i o n a d d e d t o a r e a c t i o n m i x t u r e c o n t a i n i n g l i p i d , 0.96 mg/ml ( B ) . - 98 -INCUBATION TIME , min F i g . 22. E f f e c t o f m e n a d i o n e on NADPH o x i d a t i o n . E a c h r e a c t i o n f l a s k c o n t a i n e d : 1.0 m l o f T r i s - M g C l 2 b u f f e r ; 2.0 m l o f enzyme p r e p a r a t i o n ; 0.00, 0.05, o r 6.10 ymole o f m e n a d i o n e d i s s o l v e d i n 0.1 m l o f e t h a n o l . NADPH CO.7 y m o l e ) d i s s o l v e d i n 0.1 m l o f T r i s - M g C l 2 b u f f e r was a d d e d t o e a c h r e a c t i o n f l a s k a t z e r o t i m e . C o n t r o l (©); p l u s m e n a d i o n e , 3.13 (a) o r 15.7 (A ) y m o l e s / 1 f i n a l c o n c e n t r a t i o n ; m e n a d i o n e c o n t r o l , 2 m l o f a 0.154 M KC1 s o l u t i o n i n s t e a d o f enzyme p r e p a r a t i o n was a d d e d t o a r e a c t i o n m i x t u r e c o n t a i n i n g m e n a d i o n e , 31.3 y m o l e s / 1 , f i n a l c o n c e n t r a t i o n ( A ) . - 99 -by t h e a c e t o n e powder enzyme i s a f f e c t e d b y m e n a d i o n e a nd by l i p i d e x t r a c t s i n a manner s i m i l a r t o t h e m i c r o s o m a l NADPH-c y t o c h r o m e c r e d u c t a s e d e s c r i b e d b y K a m i n et al. ( 6 2 ) . The p r e s e n c e o f m e n a d i o n e (16 -pmoles/1, f i n a l c o n c e n t r a t i o n ) p r o -d u c e s a g r e a t e r t h a n t w o - f o l d i n c r e a s e i n t h e i n i t i a l r a t e o f NADPH o x i d a t i o n ( F i g . 2 2 ) . The s p e c i f i c i t y o f t h e m e n a d i o n e - s t i m u l a t e d NADPH o x i -d a s e a c t i v i t y was e x a m i n e d a nd t h e r e s u l t s a r e shown i n F i g u r e 23. 1 , 4 - N a p h t h o q u i n o n e p r o d u c e s an e x t e n s i v e s t i m u l a t i o n o f t h e r a t e o f NADPH o x i d a t i o n w h i l e 1 , 4 - t o l u o q u i n o n e a n d 1,4-b e n z o q u i n o n e , members o f t h e coenzyme Q s e r i e s , h a v e o n l y a s l i g h t s t i m u l a t o r y e f f e c t . Of t h e two b e n z o q u i n o n e s , 1,4-t o l u o q u i n o n e i s more e f f e c t i v e , p r o d u c i n g a g r e a t e r s t i m u l a t i o n o f NADPH o x i d a t i o n t h a n 1 , 4 - b e n z o q u i n o n e a t o n e - t e n t h t h e c o n -c e n t r a t i o n o f b e n z o q u i n o n e . T h e r e f o r e t h e m e n a d i o n e r e d u c t a s e o f t h e m i t o c h o n d r i a l a c e t o n e powder e x t r a c t i s e s s e n t i a l l y s p e c i f i c f o r n a p h t h o q u i n o n e s . E r n s t e r et al. (136,137) h a v e shown t h a t t h e s o l u b l e f r a c t i o n o f r a t l i v e r c y t o p l a s m c o n t a i n s a d i c u m a r o l - s e n s i -t i v e d i a p h o r a s e c a t a l y z i n g t h e r e d u c t i o n o f v a r i o u s q u i n o n e s and d y e s w i t h e i t h e r NADH o r NADPH a s e l e c t r o n d o n o r . However t h e m i c r o s o m a l NADPH-cytochrome a r e d u c t a s e d e s c r i b e d by S a t o et al. (146,147) was i n s e n s i t i v e t o d i c u m a r o l , e v e n a t h i g h c o n c e n t r a t i o n s (0.2 mM) o f t h i s compound. The e f f e c t o f d i -c u m a r o l on t h e a c e t o n e e x t r a c t - s t i m u l a t e d a n d m e n a d i o n e - s t i m u -l a t e d NADPH o x i d a t i o n i n t h e m i t o c h o n d r i a l a c e t o n e powder enzyme p r e p a r a t i o n was t h e r e f o r e e x a m i n e d . The i n i t i a l r a t e o f NADPH o x i d a t i o n i n t h e p r e s e n c e o f a c e t o n e - e x t r a c t e d l i p i d , - 100 -INCUBATION T IME , min F i g . 23. E f f e c t of 1,4-naphthoquinone, 1,4-toluoquinone, and 1,4-benzoquinone on NADPH o x i d a t i o n . Incubations were c a r r i e d out as described i n F i g . 22. 1,4-naphthoquinone (0.1 ymole), 1,4-toluoquinone (0.1 ymole) or 1,4-benzoquinone (1.0 ymole) d i s s o l v e d i n 0.1 ml ethanol was added i n s t e a d of menadione. C o n t r o l (©); plus 1,4-naphthoquinone (o); plus 1,4-toluoquinone ( A ) ; plus 1,4-benzoquinone (•). - 101 -m e n a d i o n e , a n d d i c u m a r o l a r e shown i n T a b l e V I I . A d d i t i o n o f t h e a c e t o n e e x t r a c t ( 3 . 1 m g / m l f i n a l c o n c e n t r a t i o n ) a l o n e p r o d u c e s a f o u r - f o l d i n c r e a s e i n t h e r a t e o f NADPH o x i d a t i o n . M e n a d i o n e (31 u m o l e s / 1 ) p r o d u c e s a t w o - f o l d i n c r e a s e i n NADPH o x i d a t i o n , w h i l e d i c u m a r o l p r o d u c e s o n l y a s l i g h t s t i m u l a t i o n . When d i c u m a r o l i s a d d e d t o t h e r e a c t i o n m i x t u r e c o n t a i n i n g a c e -t o n e - e x t r a c t e d l i p i d t h e r e i s a t w o - f o l d i n c r e a s e i n t h e r a t e o f NADPH o x i d a t i o n o v e r t h a t p r o d u c e d by t h e a d d i t i o n o f t h e a c e t o n e e x t r a c t a l o n e . T h i s s t i m u l a t i o n i s e v e n m o r e p r o n o u n c e d when d i c u m a r o l i s a d d e d i n t h e p r e s e n c e o f m e n a d i o n e , r e s u l t i n g i n a s i x - f o l d i n c r e a s e i n t h e r a t e o f NADPH o x i d a t i o n o v e r t h a t p r o d u c e d b y m e n a d i o n e a l o n e . T h u s t h e o x i d a s e a c t i v i t y o f t h e m i t o c h o n d r i a l a c e t o n e p o w d e r enzyme p r e p a r a t i o n d i f f e r s f r o m b o t h t h e d i c u m a r o l - i n h i b i t e d d i a p h o r a s e d e s c r i b e d b y E r n s t e r et at. ( 1 3 6 , 1 3 7 ) a n d t h e d i c u m a r o l - i n s e n s i t i v e N A D P H - n a p h t h o -q u i n o n e r e d u c t a s e ( N A D P H - c y t o c h r o m e o r e d u c t a s e ) d e s c r i b e d b y S a t o et al. ( 1 4 6 , 1 4 7 ) . T h i s d i c u m a r o l - p r o d u c e d s t i m u l a t i o n o f NADPH o x i d a t i o n i n t h e p r e s e n c e o f m e n a d i o n e ( T a b l e V I I ) i s e x t r e m e l y s e n s i t i v e t o d i c u m a r o l a n d i s o b s e r v e d a t d i c u m a r o l — 8 c o n c e n t r a t i o n s a s l ow a s 1 .6 x 10 M . The e n h a n c i n g e f f e c t o f d i c u m a r o l o n b o t h t h e m e n a d i o n e - s t i m u l a t e d a n d a c e t o n e - e x -t r a c t e d l i p i d - s t i m u l a t e d o x i d a t i o n o f NADPH s u g g e s t s t h e p o s s i -b i l i t y t h a t f a c t o r s i n t h e l i p i d e x t r a c t r e s p o n s i b l e f o r t h i s s t i m u l a t i o n may be c o m p o u n d s a n a l o g o u s t o m e n a d i o n e . (b) E f f e c t o f A c e t o n e - E x t r a c t e d L i p i d a n d M e n a d i o n e on l l g - H y d r o x y l a t i o n H a v i n g e s t a b l i s h e d t h e e f f e c t s o f t h e a c e t o n e - e x t r a c t e d - 102 ~ T A B L E V I I E f f e c t o f m i t o c h o n d r i a l a c e t o n e - e x t r a c t e d l i p i d , m e n a d i o n e , a n d d i c u m a r o l o n t h e i n i t i a l r a t e o f NADPH o x i d a t i o n A d d i t i o n s I n i t i a l r a t e o f NADPH o x i d a t i o n A A b s o r b a n c e (340 my) p e r m i n u t e None 0. 021 L i p i d ( 3 . 1 m g / m l ) 0 . 084 M e n a d i o n e (31 y m o l e s / 1 ) 0 . 049 D i c u m a r o l (155 y m o l e s / 1 ) 0 . 030 L i p i d p l u s d i c u m a r o l 0 . 164 M e n a d i o n e p l u s d i c u m a r o l 0 . 290 A p p r o p r i a t e r e a c t i o n f l a s k s c o n t a i n e d : 1 .0 m l o f T r i s -M g C l 2 b u f f e r ; 2 . 0 m l o f enzyme p r e p a r a t i o n ; l i p i d (10 mg) s u s -p e n d e d i n 0 . 1 m l o f T r i s-MgCl2 b u f f e r ; m e n a d i o n e ( 0 . 1 y m o l e ) . d i s s o l v e d i n 0 . 0 2 m l o f e t h a n o l , d i c u m a r o l ( 0 . 5 y m o l e ) d i s s o l v e d i n 0 . 0 5 m l o f a d i l u t e a l c o h o l i c KOH s o l u t i o n . T h e a p p r o p r i a t e s o l v e n t was a d d e d t o r e a c t i o n f l a s k s n o t r e c e i v i n g l i p i d , m e n a -d i o n e , o r d i c u m a r o l . NADPH ( 0 . 8 y m o l e ) d i s s o l v e d i n 0 . 0 5 m l o f T r i s - M g C l 2 b u f f e r was a d d e d a t z e r o t i m e . - 103 -l i p i d a n d m e n a d i o n e o n NADPH o x i d a t i o n , i t was deemed i m p o r -t a n t t o d e t e r m i n e w h e t h e r o r n o t s i m i l a r e f f e c t s on 113-hydroxy-l a t i o n w e r e e x e r t e d by t h e s e f a c t o r s . The a c e t o n e - e x t r a c t e d l i p i d f r a c t i o n a n d m e n a d i o n e w e r e e x a m i n e d f o r t h e i r . e f f e c t s on t h e NADPH o x i d a t i o n a s s o c i a t e d w i t h t h e h y d r o x y l a t i o n o f s u b s t r a t e d e o x y c o r t i c o s t e r o n e . The e f f e c t o f t h e a c e t o n e - e x t r a c t e d l i p i d f r a c t i o n i s shown i n F i g u r e 24. The a d d i t i o n o f s u b s t r a t e d e o x y c o r t i c o s t e r o n e (186 y m o l e s / 1 , f i n a l c o n c e n t r a t i o n ) i n c r e a s e s t h e i n i t i a l r a t e o f NADPH o x i d a t i o n f r o m 10 m y m o l e s / m i n t o 15 m y m o l e s / m i n . A d d i t i o n o f a c e t o n e - e x t r a c t e d l i p i d i n c r e a s e s t h e i n i t i a l r a t e o f NADPH o x i d a t i o n f r o m 10 m y m o l e s / m i n t o 45 m y m o l e s / m i n . A d d i t i o n o f d e o x y c o r t i c o s t e r o n e i n t h e p r e s e n c e o f l i p i d r e s u l t s i n a f u r t h e r s t i m u l a t i o n o f NADPH o x i d a t i o n , t o 58 m y m o l e s / m i n . Thus t h e i n c r e a s e i n t h e r a t e o f NADPH o x i -d a t i o n p r o d u c e d by d e o x y c o r t i c o s t e r o n e i n t h e p r e s e n c e o f t h e a c e t o n e - e x t r a c t (13 mymoles/min) i s g r e a t e r t h a n t h a t p r o d u c e d b y t h e d e o x y c o r t i c o s t e r o n e i n t h e a b s e n c e o f t h i s l i p i d (5 my m o l e s / m i n ) a n d s u g g e s t s t h a t t h e l i p i d e x t r a c t i s i n c r e a s i n g t h e r a t e o f 1 1 3 - h y d r o x y l a t i o n . NADPH o x i d a t i o n i n t h e p r e s e n c e o f d i c u m a r o l i s n o t a f f e c 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 d e o x y c o r t i c o s t e r o n e ( F i g . 24). T h e r e f o r e d i c u m a r o l i n h i b i t s t h e i n c r e a s e i n NADPH o x i d a t i o n t h a t i s n o r m a l l y p r o d u c e d b y t h e a d d i t i o n o f d e o x y c o r t i c o s t e r o n e ( F i g s . 4 a n d 24), a f i n d -i n g i n a g r e e m e n t w i t h t h e e s t a b l i s h e d i n h i b i t o r y a c t i o n o f d i c u m a r o l o n 1 1 3 - h y d r o x y l a t i o n . T h i s i n h i b i t i o n o f d e o x y -c o r t i c o s t e r o n e - a s s o c i a t e d NADPH o x i d a t i o n i s n o t o v e r c o m e by t h e a d d i t i o n o f t h e a c e t o n e - e x t r a c t e d l i p i d f r a c t i o n ( F i g . - 1 0 4 -1.6 INCUBATION TIME.min F i g . 24. E f f e c t o f d e o x y c o r t i c o s t e r o n e , m i t o c h o n d r i a l a c e t o n e -e x t r a c t e d l i p i d , a n d d i c u m a r o l on NADPH o x i d a t i o n . A p p r o p r i a t e r e a c t i o n f l a s k s c o n t a i n e d : d e o x y c o r t i c o -s t e r o n e (186 u m o l e s / 1 , f i n a l c o n c e n t r a t i o n ) d i s s o l v e d i n 0.05 m l o f p r o p y l e n e g l y c o l ; 1.0 m l o f T r i s - M g C l 2 b u f f e r ; 2.0 m l o f enzyme p r e p a r a t i o n ; a c e t o n e - e x t r a c t e d l i p i d (10 mg) s u s -p e n d e d i n 0.1 m l o f T r i s - M g C l 2 b u f f e r ; a n d d i c u m a r o l (0.5 umole) d i s s o l v e d i n 0.05 ml o f a d i l u t e a l c o h o l i c KOH s o l u t i o n . The a p p r o p r i a t e s o l v e n t was a d d e d t o r e a c t i o n f l a s k s n o t r e c e i v i n g d e o x y c o r t i c o s t e r o n e , l i p i d , o r d i c u m a r o l . NADPH (0.8 umole) d i s s o l v e d i n 0.05 m l o f T r i s - M g C l 2 b u f f e r was a d d e d a t z e r o t i m e . C o n t r o l (o O) ; p l u s d e o x y c o r t i c o s t e r o n e [& o) ; p l u s d i c u m a r o l (• • ) ; p l u s d i c u m a r o l a n d d e o x y c o r t i c o s t e r o n e ( B a); p l u s l i p i d ( A A ) ; p l u s l i p i d a n d d e o x y c o r t i c o s t e r o n e ( A A ) ; p l u s l i p i d and d i c u m a r o l (© © ) ; p l u s l i p i d , d i c u m a r -o l a n d d e o x y c o r t i c o s t e r o n e (O o ) . - 105 -24) . The r a p i d o x i d a t i o n o f NADPH i n t h e p r e s e n c e o f b o t h d i c u m a r o l a n d t h e a c e t o n e - e x t r a c t e d l i p i d i s n o t a l t e r e d upon a d d i t i o n o f d e o x y c o r t i c o s t e r o n e . The e f f e c t o f m e n a d i o n e on NADPH o x i d a t i o n a s s o c i a t e d w i t h 1 1 3 - h y d r o x y l a t i o n i s shown i n F i g u r e 25. I n t h i s i n -s t a n c e t h e i n c r e a s e i n NADPH o x i d a t i o n p r o d u c e d b y t h e a d d i -t i o n o f d e o x y c o r t i c o s t e r o n e i n t h e p r e s e n c e o f m e n a d i o n e i s no g r e a t e r t h a n t h a t r e s u l t i n g f r o m t h e a d d i t i o n o f d e o x y -c o r t i c o s t e r o n e i n t h e a b s e n c e o f m e n a d i o n e . T h e r e f o r e i t a p p e a r s t h a t m e n a d i o n e i s n o t i n c r e a s i n g t h e r a t e o f l l g -h y d r o x y l a t i o n . The e x t e n s i v e s t i m u l a t i o n o f NADPH o x i d a -t i o n p r o d u c e d b y t h e s i m u l t a n e o u s a d d i t i o n o f m e n a d i o n e a n d d i c u m a r o l o c c u r s i n b o t h t h e a b s e n c e a nd p r e s e n c e o f s t e r o i d s u b s t r a t e . W h i l e t h e e f f e c t s o f b o t h t h e a c e t o n e - e x t r a c t e d l i p i d f r a c t i o n a n d m e n a d i o n e on " b a c k g r o u n d " NADPH o x i d a t i o n i n b o t h t h e a b s e n c e a n d p r e s e n c e o f d i c u m a r o l a r e s i m i l a r , t h e a c e t o n e - e x t r a c t e d l i p i d f r a c t i o n p o s s e s s e s a f a c t o r ( o r f a c t o r s ) t h a t i n c r e a s e s t h e r a t e o f 1 1 3 - h y d r o x y l a t i o n . A more c r i t i c a l e x a m i n a t i o n o f t h e e f f e c t s o f m e n a d i o n e a n d t h e a c e t o n e - e x t r a c t e d l i p i d m a t e r i a l on 1 1 3 - h y d r o x y l a t i o n was a c c o m p l i s h e d by d i r e c t m e a s u r e m e n t o f c o r t i c o s t e r o n e f o r m a t i o n . The r e s u l t s o f t h e s e s t u d i e s a r e shown i n F i g u r e 26. I n t h e p r e s e n c e o f t h e a c e t o n e - e x t r a c t e d l i p i d f r a c t i o n t h e r e i s a s t i m u l a t i o n o f t h e r a t e o f c o r t i c o s t e r o n e f o r m a -t i o n , r e s u l t i n g i n a 27% i n c r e a s e i n t h e amount o f c o r t i c o -s t e r o n e f o r m e d a f t e r 8 m i n u t e s o f i n c u b a t i o n . T h i s f i n d i n g i s i n a g r e e m e n t w i t h t h e s t i m u l a t i o n o f d e o x y c o r t i c o s t e r o n e -a s s o c i a t e d NADPH o x i d a t i o n shown i n F i g u r e 24. S i m i l a r r e -- 106 -INCUBATION TIME,min F i g . 25. E f f e c t of de o x y c o r t i c o s t e r o n e , menadione and dicumarol on NADPH o x i d a t i o n . Incubations were c a r r i e d out as described i n F i g . 24. Menadione (0.1 ymole) d i s s o l v e d i n 0.02 ml of ethanol was added i n s t e a d of the m i t o c h o n d r i a l acetone-extracted l i p i d . C o n t r o l (o); plus deoxycorticosterone (©); plus menadione (•) ; plus menadione and deoxycorticosterone (ta) ; plus menadione and dicumarol (A); plus menadione, de o x y c o r t i c o s t e r o n e , and dicumarol (A.) . - 107 -INCUBATION TIME , min F i g . 2 6 . E f f e c t o f m i t o c h o n d r i a l a c e t o n e - e x t r a c t e d l i p i d , m e n a d i o n e , a n d d i c u m a r o l o n t h e l l B - h y d r o x y l a t i o n o f d e o x y -c o r t i c o s t e r o n e . I n c u b a t i o n s 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 i n F i g s . 24 a n d 2 5 . D e o x y c o r t i c o s t e r o n e - 1 , 2 - 3 H ( s p e c i f i c a c t i v i t y 1 . 0 7 x 1 0 6 c p m / y m o l e ) was a d d e d d i s s o l v e d i n 0 . 0 5 m l o f p r o p y l e n e g l y c o l t o e a c h r e a c t i o n f l a s k t o g i v e a f i n a l c o n c e n t r a t i o n o f 197 y m o l e s / 1 . NADPH ( 2 . 2 y m o l e s ) d i s s o l v e d i n 0 . 0 8 . m l o f T r i s - M g C l 2 b u f f e r was a d d e d a t z e r o t i m e . A f t e r 8 , 1 6 , a n d 24 m i n u t e s o f i n c u b a t i o n , a s a m p l e ( 1 . 0 ml ) was r e m o v e d f r o m e a c h r e a c t i o n f l a s k a n d p i p e t t e d i n t o e t h y l a c e t a t e (2 ml) t o t e r m i n a t e t h e r e a c t i o n . C o n t r o l ( O ) ; p l u s l i p i d ( A ) ; p l u s m e n a d i o n e ( a ) ; p l u s d i -c u m a r o l ( © ) ; p l u s l i p i d a n d d i c u m a r o l ( A ) ; p l u s m e n a d i o n e a n d d i c u m a r o l ( • ) . - 108 -s u i t s h a v e b e e n r e p o r t e d by I m a i and S a t o (148,149) who f o u n d t h a t t h e a r o m a t i c h y d r o x y l a t i o n o f a n i l i n e by an a c e t o n e powder o f r a b b i t - l i v e r m i c r o s o m e s was d e p e n d e n t u pon a l i p i d f r a c t i o n e x t r a c t e d f r o m t h e s e m i c r o s o m e s by t r e a t m e n t w i t h an a c e t o n e - m e t h a n o l - e t h e r m i x t u r e . The i n h i b i t i o n o f 1 1 3 - h y d r o x y -l a t i o n by d i c u m a r o l (62% a f t e r 8 m i n u t e s o f i n c u b a t i o n ) i s o n l y s l i g h t l y r e l i e v e d b y t h e a d d i t i o n o f t h e a c e t o n e - e x t r a c t e d l i p i d f r a c t i o n . M e n a d i o n e p r o d u c e s no i n c r e a s e i n c o r t i c o s t e r o n e f o r m a t i o n ; i n d e e d on p r o l o n g e d i n c u b a t i o n , t h e r e i s a d e c r e a s e i n t h e r a t e o f 1 1 3 - h y d r o x y l a t i o n i n t h e p r e s e n c e o f m e n a d i o n e when c o m p a r e d t o t h e c o n t r o l r a t e ( F i g . 26). T h i s d e c r e a s e i n t h e r a t e o f 1 1 3 - h y d r o x y l a t i o n i s l i k e l y due t o t h e i n c r e a s e d u t i l i z a t i o n o f NADPH i n t h e p r e s e n c e o f m e n a d i o n e ( F i g . 25). Thus u n d e r c o n d i t i o n s o f l i m i t e d a v a i l a b i l i t y o f NADPH, m e n a d i o n e becomes an i n h i b i t o r o f 1 1 3 - h y d r o x y l a t i o n due t o c o m p e t i t i o n w i t h t h e 1 1 3 - h y d r o x y l a s e f o r NADPH. T h i s i s e v i d e n t f r o m t h e h i g h d e g r e e o f i n h i b i t i o n o f 1 1 3 - h y d r o x y l a t i o n o b t a i n e d u p o n a d d i t i o n o f b o t h m e n a d i o n e a nd d i c u m a r o l ( F i g . 26), c a u s e d by t h e e x t r e m e l y r a p i d u t i l i z a t i o n o f NADPH ( F i g . 25). (c) A P o s s i b l e S i t e f o r M e n a d i o n e R e d u c t i o n I n r e c e n t s t u d i e s o n t h e e l e c t r o n - t r a n s p o r t s y s t e m i n -v o l v e d i n 1 1 3 - h y d r o x y l a t i o n , K i m u r a (60) h a s o b s e r v e d t h a t f e r r i c y a n i d e , m e n a d i o n e , o r c y t o c h r o m e c, as w e l l as t h e n a t u r a l a c c e p t o r , a d r e n o d o x i n , c a n a c t as e l e c t r o n a c c e p t o r s f o r t h e o x i d a t i o n o f NADPH by a d r e n o d o x i n r e d u c t a s e . Of - 109 -t h e e l e c t r o n a c c e p t o r s t e s t e d b y K i m u r a , f e r r i c y a n i d e was t h e m o s t e f f e c t i v e . M a x i m a l a d r e n o d o x i n r e d u c t a s e a c t i v i t y i n t h e p r e s e n c e o f m e n a d i o n e a n d c y t o c h r o m e c was r e s p e c -t i v e l y 4 1 % and 7% o f t h a t i n t h e p r e s e n c e o f a d r e n o d o x i n . K i m u r a a n d S u z u k i (59) h a v e r e p o r t e d t h a t c y t o c h r o m e P-450, c y t o c h r o m e P-420, a nd t h e f l a v o p r o t e i n a d r e n o d o x i n r e d u c t a s e o f t h e l l g - h y d r o x y l a s e e x t r a c t e d f r o m a n a d r e n a l m i t o c h o n d r i a • a c e t o n e powder a r e a l l p r e s e n t i n a f r a c t i o n p r e c i p i t a t i n g b e t w e e n 20% and 40% s a t u r a t i o n w i t h ammonium s u l f a t e . T h e s e f i n d i n g s h a v e b e e n c o n f i r m e d i n t h e p r e s e n t s t u d i e s ( F i g s . 10a and 1 0 b ) . T h i s ammonium s u l f a t e f r a c t i o n i s e s s e n t i a l l y d e v o i d o f a d r e n o d o x i n w h i c h p r e c i p i t a t e s a t h i g h e r ammonium s u l f a t e c o n c e n t r a t i o n s ( 5 9 ) . NADPH o x i d a -t i o n by t h e 20- 4 0 % ammonium s u l f a t e f r a c t i o n , a n d t h e e f f e c t o f m e n a d i o n e upon t h i s o x i d a t i o n w e r e t h e r e f o r e e x a m i n e d . NADPH o x i d a t i o n b y t h e f r a c t i o n s p r e c i p i t a t i n g b e t w e e n 20% a n d 4 0 % , and 40% and 80% s a t u r a t i o n w i t h ammonium s u l -f a t e i s shown i n T a b l e V I I I . The 20-40% ammonium s u l f a t e f r a c t i o n o x i d i z e s NADPH a t a s l o w r a t e . D e t e r m i n a t i o n o f t h e c a r b o n m o n o x i d e d i f f e r e n c e s p e c t r u m o f t h i s f r a c t i o n i n t h e p r e s e n c e o f NADPH i n d i c a t e s t h a t t h e s l o w o x i d a t i o n o f NADPH i s a c c o m p a n i e d b y a s m a l l d e g r e e o f r e d u c t i o n o f b o t h c y t o c h r o m e P-450 (A a b s o r b a n c e 450-500 my) and c y t o c h r o m e P-420 (A a b s o r b a n c e 420-500 my). R e d u c t i o n o f t h e t o t a l h e m o p r o t e i n c o n t e n t o f t h e 20-40% ammonium s u l f a t e f r a c t i o n w i t h d i t h i o n i t e i n d i c a t e s t h a t o n l y a s m a l l p o r t i o n o f t h e t o t a l c y t o c h r o m e s p r e s e n t i s r e d u c e d by NADPH. T h i s s l o w T A B L E V I I I T h e r e l a t i o n s h i p b e t w e e n o x i d a t i o n o f NADPH a n d r e d u c t i o n o f c y t o c h r o m e s P - 4 50 a n d P - 4 2 0 i n t h e 20-40% a n d 40-80% ammonium s u l f a t e f r a c t i o n s o f t h e m i t o c h o n d r i a l a c e t o n e powder enzyme p r e p a r a t i o n ( N H i t ) 2 S 0 4 f r a c t i o n NADPH NADPH-• r e d u c e d D i t h i o n i t e - r e d u c e d i n c u b a t e d o x i d a t i o n P - 4 5 0 & P - 4 2 0 P - 4 5 0 & P - 4 2 0 A A b s o r b a n c e (340 mu) A A b s o r b a n c e A A b s o r b a n c e p e r m i n u t e 450 -500 4 2 0 - 5 0 0 4 5 0 - 5 0 0 4 2 0 - 5 0 0 my my my my 2 0 - 40% (no NADPH a d d e d ) 0 . 0 0 0 0 . 0 0 0 0 . 0 0 4 0 . 0 4 5 0 . 0 6 7 2 0 - 40% 0 . 0 1 5 0 . 0 0 8 0 . 0 1 3 0 . 0 4 4 0 . 0 5 3 4 0 - 80% ( 0 . 1 ml) 0 . 0 1 0 0 . 0 0 0 0 . 1 2 0 . 0 0 0 0 . 2 0 4 0 - 80% ( 0 . 2 ml) 0 . 0 2 2 0 . 0 0 0 0 . 2 4 0 . 0 0 0 0 . 3 4 (20 -40%) + 0 . 1 m l (40-80%) 0 . 0 3 5 0 . 0 0 4 0 . 1 2 0 . 0 4 2 0. 22 (20 -40%) + 0 . 2 m l (40-80%) 0. 060 0 . 0 0 6 0.26 0 . 0 3 8 0.3.4 The 20-40% a n d 40-80% ammonium s u l f a t e f r a c t i o n s o b t a i n e d f r o m 4 m l o f t h e e n -zyme p r e p a r a t i o n w e r e e a c h d i s s o l v e d i n a s o l u t i o n made up o f T r i s - M g C l 2 b u f f e r ( 0 . 4 2 ml) a n d 0 . 1 5 4 M K C l ( 0 . 7 4 m l ) . A p p r o p r i a t e c u v e t t e s c o n t a i n e d : .0.58 m l o f t h e 20-40% f r a c t i o n ; 0 . 1 o r 0 . 2 m l o f t h e 40-80% f r a c t i o n ; 0 . 0 1 m l o f e t h a n o l , - a n d 0 . 0 2 m l o f p r o p y l e n e g l y c o l . T h e v o l u m e o f t h e r e a c t i o n m i x t u r e was a d j u s t e d t o 0 . 8 1 m l w i t h t h e T r i s - K C l s o l u t i o n . NADPH o x i d a t i o n was m e a s u r e d by a d d i t i o n o f NADPH ( 0 . 2 7 ymole ) t o t h e s a m p l e c u v e t t e . R e d u c t i o n o f c y t o c h r o m e s P - 4 5 0 a n d P - 4 2 0 was m e a s u r e d ( a f t e r a d d i t i o n o f NADPH ( 0 . 2 7 y m o l e ) o r d i t h i o n i t e t o b o t h t h e s a m p l e a n d r e f e r e n c e c u -v e t t e s ) b y d e t e r m i n i n g t h e c a r b o n m o n o x i d e d i f f e r e n c e s p e c t r u m o f t h e r e a c t i o n m i x t u r e . - I l l -r a t e o f NADPH o x i d a t i o n a n d l i m i t e d r e d u c t i o n o f c y t o c h r o m e s P-450 a n d P-420 a r e e x p e c t e d r e s u l t s b e c a u s e a d r e n o d o x i n , t h e component o f t h e 1 1 3 - h y d r o x y l a s e p a t h w a y w h i c h f u n c t i o n s i n t h e t r a n s f e r o f e l e c t r o n s f r o m a d r e n o d o x i n r e d u c t a s e t o c y t o -chrome P-450, i s n o t p r e s e n t i n t h i s ammonium s u l f a t e f r a c t i o n . The s m a l l amounts o f c y t o c h r o m e P-450 a n d c y t o c h r o m e P-420 t h a t a r e r e d u c e d may i n d i c a t e t h e p r e s e n c e o f t r a c e amounts o f a d r e n o d o x i n i n t h i s f r a c t i o n . The 4 0-80% ammonium s u l f a t e f r a c t i o n a l s o c a t a l y z e s a s l o w r a t e o f NADPH o x i d a t i o n ( T a b l e V I I I ) , p e r h a p s v i a some o t h e r f l a v o p r o t e i n t h a t i s p r e s e n t i n t h i s f r a c t i o n ( F i g . 1 1 a ) . R e c o m b i n a t i o n o f t h e 20-4 0 % a nd 40-80% f r a c t i o n r e -s u l t s i n a n i n c r e a s e i n NADPH o x i d a t i o n , t h e i n c r e a s e d r a t e b e i n g g r e a t e r t h a n t h e sum o f t h e NADPH o x i d i z e d by b o t h f r a c t i o n s a l o n e . U n f o r t u n a t e l y a t t e m p t s t o show t h a t t h i s i n c r e a s e d o x i d a t i o n o f NADPH i s a c c o m p a n i e d by a n i n c r e a s e d r e d u c t i o n o f c y t o c h r o m e P-450 w e r e u n s u c c e s s f u l b e c a u s e t h e 40-80% f r a c t i o n c o n t a i n e d m a t e r i a l w h i c h b o u n d c a r b o n m o n o x i d e and e x h i b i t e d an i n t e n s e a b s o r p t i o n maximum a t 4 20 my i n t e r -f e r i n g w i t h t h e m e a s u r e m e n t o f c y t o c h r o m e P-450 ( T a b l e V I I I ) . H o w ever, K i m u r a (60) h a s shown t h a t a d r e n o d o x i n i s r e q u i r e d f o r r e d u c t i o n o f c y t o c h r o m e P-4 20. The e f f e c t o f m e n a d i o n e on NADPH o x i d a t i o n was n e x t e x a m i n e d . ( F i g . 2 7 a ) . The p r e s e n c e o f m e n a d i o n e (72 y m o l e s / 1 , f i n a l c o n c e n t r a t i o n ) i n c r e a s e s t h e r a t e o f o x i d a t i o n o f NADPH f r o m 2.8 m y m o l e s / m i n t o 59 m y m o l e s / m i n , a 2 1 - f o l d i n -c r e a s e . I t i s t h e r e f o r e e v i d e n t t h a t m e n a d i o n e c a n a c t as - 112 -.0 8 . 0 4 LU O ? . 0 2 CO cc o 1 0 C O < 0 - . 0 2 5 0 0 4 8 0 4 6 0 4 4 0 4 2 0 4 0 0 W A V E L E N G T H , mji F i g . 2 7 a . E f f e c t o f m e n a d i o n e on t h e o x i d a t i o n o f NADPH b y t h e 20-40% ammonium s u l f a t e f r a c t i o n o f t h e m i t o c h o n d r i a l a c e t o n e powder, enzyme p r e p a r a t i o n . T h e 20-40% ammonium s u l f a t e p r e c i p i t a t e o b t a i n e d f r o m 4 m l o f enzyme p r e p a r a t i o n was d i s s o l v e d i n a s o l u t i o n made up o f T r i s - M g C l 2 b u f f e r ( 0 . 6 ml) a n d 0 . 1 5 4 M KC1 ( 1 . 0 m l ) . P r o p y -l e n e g l y c o l ( 0 . 0 4 m l ) , a n d m e n a d i o n e (72 y m o l e s / 1 , f i n a l c o n -c e n t r a t i o n ) d i s s o l v e d i n 0 . 0 2 m l o f e t h a n o l w e r e a d d e d a n d t h e m i x t u r e was d i v i d e d e q u a l l y b e t w e e n two c u v e t t e s . A f t e r p r e -i n c u b a t i o n a t 2 5 ° C f o r 6 m i n u t e s , NADPH ( 0 . 2 7 y m o l e ) d i s s o l v e d i n 0 . 0 1 m l o f T r i s - M g C l 2 b u f f e r was a d d e d t o t h e s a m p l e c u v e t t e . T h e c o n t r o l i n c u b a t i o n r e c e i v e d 0 . 0 1 m l o f e t h a n o l i n s t e a d o f m e n a d i o n e . C o n t r o l ( ) ; p l u s m e n a d i o n e ( ) . F i g . 2 7 b . E f f e c t o f m e n a d i o n e o n t h e NADPH m e d i a t e d r e d u c -t i o n o f c y t o c h r o m e s P - 4 5 0 a n d P - 4 2 0 i n t h e 20-40% ammonium s u l f a t e f r a c t i o n o f t h e m i t o c h o n d r i a l a c e t o n e p o w d e r enzyme p r e p a r a t i o n . T h e r e a c t i o n m i x t u r e i n e a c h c u v e t t e was a s d e s c r i b e d i n F i g . 27a e x c e p t t h a t NADPH ( 0 . 2 7 y m o l e ) d i s s o l v e d i n 0 . 0 1 m l o f T r i s - M g C l 2 b u f f e r was a d d e d t o b o t h t h e s a m p l e a n d r e f e r e n c e c u v e t t e s . A f t e r 3 m i n u t e s o f i n c u b a t i o n , c a r b o n m o n o x i d e was b u b b l e d t h r o u g h t h e s a m p l e c u v e t t e s o l u t i o n f o r 45 s e c o n d s a n d t h e c a r b o n m o n o x i d e d i f f e r e n c e s p e c t r u m was r e c o r d e d . C o n t r o l ( ) ; p l u s m e n a d i o n e ( —) . - 113 -a n e l e c t r o n a c c e p t o r i n t h e 20-40% ammonium s u l f a t e f r a c t i o n , a f i n d i n g i n a g r e e m e n t w i t h t h a t o f K i m u r a ( 6 0 ) . The e f f e c t o f t h i s s t i m u l a t i o n o f NADPH o x i d a t i o n by m e n a d i o n e on t h e r e d u c t i o n o f c y t o c h r o m e s P-450 and P-420 i s shown i n F i g u r e 27b. T h e r e i s a s l i g h t d e c r e a s e i n t h e e x t e n t o f c y t o c h r o m e P-4 50 r e d u c t i o n on a d d i t i o n o f m e n a d i o n e . H o w e v e r , m e n a d i o n e a d d i t i o n p r o d u c e s a s u b s t a n t i a l i n c r e a s e i n c y t o c h r o m e P-420 r e d u c t i o n as i n d i c a t e d by t h e g r e a t e r t h a n t w o - f o l d i n c r e a s e i n t h e a b s o r p t i o n maximum a t 420 my. T h e r e f o r e , c y t o c h r o m e P-420 i s c a p a b l e o f a c t i n g a s a n e l e c t r o n a c c e p t o r i n t h e p r e s e n c e o f m e n a d i o n e . I n t h e 20-40% ammonium s u l f a t e f r a c -t i o n m e n a d i o n e i s , i n some manner, m e d i a t i n g t h e N A D P H - l i n k e d r e d u c t i o n o f c y t o c h r o m e P-420 by a d r e n o d o x i n r e d u c t a s e , p e r -h a p s a c t i n g as a n i n t e r m e d i a t e e l e c t r o n a c c e p t o r . N i s h i b a y a s h i et al. h a v e o b s e r v e d (14 7) t h a t t h e N A D P H - n a p h t h o q u i n o n e r e -d u c t a s e o f r a b b i t l i v e r m i c r o s o m e s c o u l d n o t u t i l i z e c y t o -chrome P-420 a s a n e l e c t r o n a c c e p t o r . T h i s i s a l s o t r u e w i t h t h e s y s t e m f r o m a d r e n a l m i t o c h o n d r i a . V e r y l i t t l e o x i d a t i o n o f NADPH o r r e d u c t i o n o f c y t o c h r o m e P-420 t a k e s p l a c e when b o t h t h e NADPH r e d u c t a s e a n d c y t o c h r o m e P-420 a r e p r e s e n t i n t h e 2 0 - 4 0 % ammonium s u l f a t e f r a c t i o n ( T a b l e V I I I , F i g . 2 7 b ) . However t h e p r e s e n c e o f m e n a d i o n e a l l o w s t h e t r a n s f e r o f e l e c t r o n s f r o m NADPH t o c y t o c h r o m e P-420 i n t h e s y s t e m e x -t r a c t e d f r o m a n a d r e n a l m i t o c h o n d r i a l a c e t o n e p o wder. (d) E x a m i n a t i o n o f t h e A c e t o n e - E x t r a c t e d L i p i d F a c t o r s  A f f e c t i n g NADPH O x i d a t i o n a n d l l g - H y d r o x y l a t i o n S t u d i e s w i t h t h e a c e t o n e - e x t r a c t e d l i p i d f r a c t i o n showed - 114 -t h a t i t a f f e c t e d b o t h NADPH o x i d a t i o n a n d l l g - h y d r o x y l a t i o n ( F i g s . 21 a n d 2 6 ) . The e f f e c t o f t h e a c e t o n e - e x t r a c t e d l i p i d f r a c t i o n o n NADPH o x i d a t i o n was v e r y s i m i l a r t o t h e e f f e c t p r o d u c e d b y m e n a d i o n e ( F i g . 2 2 ) . However, m e n a d i o n e p r o d u c e d no s t i m u l a t i o n o f l l g - h y d r o x y l a t i o n . A p r e l i m i n a r y f r a c t i o n -a t i o n o f t h e a c e t o n e - e x t r a c t e d l i p i d was u n d e r t a k e n i n a n a t t e m p t t o r e s o l v e a n d p u r i f y t h e f a c t o r s o r c o m p o n e n t s r e s p o n -s i b l e f o r t h e e f f e c t s on NADPH o x i d a t i o n a n d l l g - h y d r o x y l a t i o n . W a t e r was r e m o v e d f r o m t h e a c e t o n e - e x t r a c t e d l i p i d f r a c -t i o n by a z e o t r o p i c e v a p o r a t i o n w i t h a b s o l u t e e t h a n o l . The d r i e d m a t e r i a l was g r o u n d t o a f i n e powder a nd e x t r a c t e d w i t h p e t r o l e u m e t h e r . The p e t r o l e u m e t h e r f r a c t i o n was c o n c e n t r a t e d u n d e r r e d u c e d p r e s s u r e a n d t h e c o n c e n t r a t e was t h e n s t i r r e d i n t o a c e t o n e a n d m a i n t a i n e d a t -20° C o v e r n i g h t . The p h o s p h o -l i p i d f r a c t i o n was t h e n o b t a i n e d as a p r e c i p i t a t e ( 1 1 1 ) . A l l f r a c t i o n s w e r e e v a p o r a t e d a l m o s t t o d r y n e s s in vacuo , a n d r e -s u s p e n d e d i n T r i s b u f f e r b y u l t r a s o n i c t r e a t m e n t . P o r t i o n s o f e a c h f r a c t i o n w e r e t h e n e x a m i n e d f o r t h e i r e f f e c t on b o t h l l g - h y d r o x y l a t i o n a n d NADPH o x i d a t i o n . The r e s u l t s o f t h e s e s t u d i e s a r e shown i n T a b l e I X . I n o r d e r t o e x c l u d e t h e p o s s i -b i l i t y t h a t NADPH c o u l d become r a t e - l i m i t i n g a n d t h u s i n f l u e n c e a n y e f f e c t on l l g - h y d r o x y l a t i o n o f t h e f r a c t i o n s t o be t e s t e d , i n i t i a l r a t e s o f l l g - h y d r o x y l a t i o n a n d NADPH o x i d a t i o n w e r e m e a s u r e d ( T a b l e I X ) . The f a c t o r s s t i m u l a t i n g b o t h NADPH o x i -d a t i o n a n d l l g - h y d r o x y l a t i o n a r e l a r g e l y e x t r a c t e d f r o m t h e d r i e d a c e t o n e - e x t r a c t e d l i p i d f r a c t i o n w i t h p e t r o l e u m e t h e r , a l t h o u g h some s t i m u l a t o r y a c t i v i t y r e m a i n s i n t h e p e t r o l e u m TABLE IX E f f e c t o f a d r e n a l m i t o c h o n d r i a l i p i d f r a c t i o n s o n t h e l l g - h y d r o x y l a t i o n o f d e o x y -c o r t i c o s t e r o n e a n d on NADPH o x i d a t i o n F r a c t i o n a d d e d C o r t i c o s t e r o n e s y n t h e s i z e d NADPH o x i d i z e d y m o l e / l / m i n P e r c e n t c o n t r o l a c t i v i t y A A b s o r b a n c e a t 34 0 my p e r m i n u t e P e r c e n t c o n t r o l a c t i v i t y C o n t r o l (no a d d i t i o n s ) 0 . 4 2 3 100 0 . 0 5 100 T o t a l m i t o c h o n d r i a l l i p i d 0 . 5 7 4 136 0 . 7 0 1400 P e t r o l e u m e t h e r i n s o l u b l e 0 . 4 5 6 108 0 . 2 4 480 - 2 0 ° C A c e t o n e i n s o l u b l e 0 . 5 5 2 130 1 .58 3160 - 2 0 ° C A c e t o n e s o l u b l e 0 . 4 3 5 103 1 .26 2520 A s o l e c t i n 0 . 4 8 5 115 0 . 0 9 180 E a c h f r a c t i o n was s u s p e n d e d i n T r i s - M g C l 2 b u f f e r a t a c o n c e n t r a t i o n o f 10 mg l i p i d / m l . 1) l l g - h y d r o x y l a t i o n o f d e o x y c o r t i c o s t e r o n e . E a c h r e a c t i o n f l a s k c o n t a i n e d : d e o x y -c o r t i c o s t e r o n e - ! ^ - ^ ( s p e c i f i c a c t i v i t y 6 . 7 4 x 1 0 7 c p m / y m o l e ) d i s s o l v e d i n 0 . 0 5 m l o f p r o p y l e n e g l y c o l t o g i v e a f i n a l c o n c e n t r a t i o n o f 1 2 . 8 y m o l e s / 1 ; 0 . 5 5 m l o f T r i s -M g C l 2 b u f f e r ; 1 .3 m l o f enzyme p r e p a r a t i o n ; a n d 0 .2 m l o f t h e a p p r o p r i a t e l i p i d f r a c -t i o n . A f t e r p r e - i n c u b a t i o n f o r 8 m i n u t e s , NADPH ( 2 . 2 y m o l e s ) was a d d e d d i s s o l v e d i n 0 . 0 5 m l o f T r i s - M g C l 2 b u f f e r . A s a m p l e ( 1 . 0 ml) was r e m o v e d f r o m e a c h r e a c t i o n f l a s k a f t e r 90 s e c o n d s o f i n c u b a t i o n a n d p i p e t t e d i n t o e t h y l a c e t a t e (2 ml) t o t e r m i n a t e t h e r e a c t i o n . 2) NADPH o x i d a t i o n . E a c h r e a c t i o n f l a s k c o n t a i n e d : 0 . 0 5 m l o f p r o p y l e n e g l y c o l ; 0 . 9 m l o f T r i s - M g C l 2 b u f f e r ; 1 .95 m l o f enzyme p r e p a r a t i o n ; a n d 0 . 2 m l o f t h e a p p r o p r i a t e l i p i d f r a c t i o n . NADPH ( 1 . 0 y m o l e ) was a d d e d d i s s o l v e d i n 0 . 1 m l o f T r i s - M g C l 2 b u f f e r a t z e r o t i m e . - 116 -e t h e r - i n s o l u b l e m a t e r i a l . The 110 - h y d r o x y l a s e s t i m u l a t i n g a c t i v i t y i s p r e c i p i t a t e d b y a c e t o n e a t -20° C s u g g e s t i n g t h a t t h e f a c t o r p r o d u c i n g t h i s s t i m u l a t i o n may b e p h o s p h o l i p i d i n n a t u r e . NADPH o x i d a t i o n s t i m u l a t i n g a c t i v i t y i s p r e s e n t i n b o t h t h e -20° C a c e t o n e s o l u b l e a n d p r e c i p i t a b l e f r a c t i o n s . T h e s e r e s u l t s ( T a b l e IX) s u g g e s t t h a t t h e f a c t o r ( s ) s t i m u l a t i n g 1 1 3 - h y d r o x y l a t i o n c a n b e p a r t i a l l y r e s o l v e d f r o m t h e f a c t o r ( s ) s t i m u l a t i n g NADPH o x i d a t i o n . A l l 113 - h y d r o x y -l a s e - s t i m u l a t i n g a c t i v i t y i s c o n t a i n e d i n a f r a c t i o n p r e c i -p i t a t e d b y a c e t o n e a t -20° C , a t r e a t m e n t known t o p r e c i p i -t a t e p h o s p h o l i p i d s b u t n o t q u i n o n e s (111) . Some s t i m u l a t i o n o f 1 1 3 - h y d r o x y l a t i o n c a n be a c h i e v e d w i t h a s o l e c t i n , a c o m -m e r c i a l s o y b e a n p h o s p h o l i p i d p r e p a r a t i o n , s u p p o r t i n g t h e s u g g e s t i o n t h a t p h o s p h o l i p i d i s r e s p o n s i b l e f o r t h e s t i m u l a -t i o n o f 1 1 3 - h y d r o x y l a s e a c t i v i t y ( T a b l e I X ) . A s o l e c t i n p r o -d u c e s o n l y a n 80% i n c r e a s e i n NADPH o x i d a t i o n h o w e v e r much l e s s t h a n t h a t p r o d u c e d by t h e -20° C a c e t o n e p r e c i p i t a t e (3160%). NADPH o x i d a t i o n i s a l m o s t e q u a l l y s t i m u l a t e d b y b o t h t h e -20° C a c e t o n e s o l u b l e a n d i n s o l u b l e f r a c t i o n s . T h e s t i m u l a t i o n o f NADPH o x i d a t i o n b y t h e -20° C a c e t o n e p r e -c i p i t a t e s u g g e s t s t h a t e i t h e r t h e r e i s c o n t a m i n a t i o n o f t h i s f r a c t i o n w i t h m a t e r i a l f r o m t h e s o l u b l e f r a c t i o n , i . e . , i n -c o m p l e t e s e p a r a t i o n o f t h e s t i m u l a t i n g f a c t o r b y t h i s f r a c -t i o n a t i o n p r o c e d u r e , o r t h a t more t h a n one c o m p o n e n t i s r e -s p o n s i b l e f o r t h e s t i m u l a t i o n o f NADPH o x i d a t i o n . I n a n y - 117 -c a s e t h e s t i m u l a t i o n o f NADPH o x i d a t i o n d o e s n o t a p p e a r t o be a s s o c i a t e d w i t h t h e s t i m u l a t i o n o f 1 1 6 - h y d r o x y l a t i o n as j u d g e d b y t h e i n a b i l i t y o f t h e - 2 0 ° C a c e t o n e s o l u b l e f r a c -t i o n t o s t i m u l a t e h y d r o x y l a t i o n a n d by t h e r e l a t i v e l y l o w s t i m u l a t i o n o f NADPH o x i d a t i o n by a s o l e c t i n , w h i c h d o e s s t i m u l a t e 1 1 6 - h y d r o x y l a t i o n . F u r t h e r e x a m i n a t i o n o f t h e l i p i d s o f b e e f a d r e n a l m i t o -c h o n d r i a was u n d e r t a k e n i n a n a t t e m p t t o i s o l a t e t h e f a c t o r s t i m u l a t i n g NADPH o x i d a t i o n b y t h e enzyme p r e p a r a t i o n . T h e p r o c e d u r e u s e d was a m o d i f i c a t i o n o f t h a t e m p l o y e d b y C r a n e et al. ( I l l ) f o r t h e i s o l a t i o n o f c o e n z y m e Cho by d i r e c t s o l -v e n t e x t r a c t i o n o f b e e f h e a r t m i t o c h o n d r i a . T h e m i t o c h o n d r i a f r o m 1 0 0 0 g o f b e e f a d r e n a l g l a n d s w e r e p r o c e s s e d i n t h i s s t u d y . T h e a b i l i t y o f e a c h i s o l a t e d f r a c t i o n t o s t i m u l a t e NADPH o x i d a t i o n was m e a s u r e d a n d t h e r e s u l t s a r e shown i n T a b l e X . T h e f r a c t i o n s t h a t s t i m u l a t e NADPH o x i d a t i o n a r e known t o c o n t a i n q u i n o n e s ( 1 1 1 ) . I t i s n o t e d t h a t t h e p h o s -p h o l i p i d f r a c t i o n (-20° C a c e t o n e p r e c i p i t a t e ) d o e s n o t s t i m u l a t e NADPH o x i d a t i o n , i n d i c a t i n g a b e t t e r s e p a r a t i o n o f p h o s p h o l i p i d f r o m t h e f a c t o r s t i m u l a t i n g NADPH o x i d a t i o n t h a n was a c h i e v e d p r e v i o u s l y ( T a b l e I X ) . When t h e a c e t o n e -s o l u b l e f r a c t i o n ( T a b l e X) was e v a p o r a t e d t o d r y n e s s in vacuo a n d t h e m a t e r i a l was r e d i s s o l v e d i n e t h a n o l , w h i t e c r y s t a l s f o r m e d o n s t a n d i n g o v e r n i g h t a t - 2 0 ° C . T h e s e c r y s t a l s w e r e r e m o v e d , d r i e d , a n d t h e i r m e l t i n g p o i n t was d e t e r m i n e d a n d f o u n d t o be i d e n t i c a l w i t h a n a u t h e n t i c s a m p l e o f c h o l e s t e r o l . T h i s f r a c t i o n d o e s n o t s t i m u l a t e _ N A D P H o x i d a t i o n ( T a b l e X ) . - 118 -TABLE X E f f e c t of adrenal mitochondria l i p i d f r a c t i o n s on NADPH o x i -d a t i o n L i p i d F r a c t i o n added Percent of c o n t r o l a c t i v i t y -Incubation time (minutes) 5 15 Ethanol/ether s o l u b l e 457 261 Ethanol/ether i n s o l u b l e 100 86 Petroleum ether s o l u b l e 484 242 Petroleum ether i n s o l u b l e 111 104 -20° C Acetone s o l u b l e 320 247 -2 0° C Acetone i n s o l u b l e 106 102 -20° C Ethanol s o l u b l e 340 253 -20° C Ethanol i n s o l u b l e 103 104 Each r e a c t i o n f l a s k contained: 1.05 ml of T r i s - M g C l 2 b u f f e r ; 2.0 ml of enzyme p r e p a r a t i o n ; 0.0 5 ml of l i p i d f r a c t i o n suspended or d i s s o l v e d i n ethanol. NADPH (0.9 ymole) i n 0.1 ml of T r i s - M g C l 2 b u f f e r was added at zero time. - 119 -The f r a c t i o n a t i o n procedure o u t l i n e d i n Table X i n d i c a t e s t h a t the f a c t o r that s t i m u l a t e s NADPH o x i d a t i o n i s e x t r a c -t a b l e from mitochondria w i t h ethanol-ether, i s s o l u b l e i n petroleum ether, acetone at -20° C, and ethanol at -20° C, and i s n e i t h e r phospholipid nor c h o l e s t e r o l . The m a t e r i a l obtained from the -20° C et h a n o l - s o l u b l e f r a c t i o n was d i s s o l v e d i n a small amount of hexane and a p p l i e d t o a s i l i c a g e l column (1 cm x 19 cm). The column was developed by a m o d i f i c a t i o n of the procedures described by Crane et at. ( I l l ) and by Sottocasa and Crane (150). Each eluate was examined f o r i t s a b i l i t y to st i m u l a t e NADPH o x i -d a t i o n . The r e s u l t s are shown i n Table XI. Y e l l o w i s h m a t e r i a l i s e l u t e d by each of the solvent combinations but only those f r a c t i o n s e l u t e d w i t h 100% chloroform s t i m u l a t e the o x i d a t i o n of NADPH. Crane et aib~ (111) have reported t h a t coenzyme Q i 0 i s elu t e d from s i l i c a g e l w i t h t h i s s o l - , vent. A p o r t i o n of each column eluate was a p p l i e d to a s i l i c a g e l t h i n - l a y e r chromatography p l a t e , and the p l a t e was developed w i t h benzene. Only the three f r a c t i o n s t h a t s t i m u l a t e d NADPH o x i d a t i o n had yellow, u l t r a v i o l e t absorbing zones, which e x h i b i t e d an value corresponding to that of an aut h e n t i c sample of coenzyme Qio. These zones appeared as blUe spots when sprayed w i t h leucomethylene blue prepared by the method of Crane and D i l l e y (112). The absorption spectra of the eluates c o n t a i n i n g coenzyme Qio e x h i b i t e d a shoulder at 275 my, the regi o n of maximum ab-s o r p t i o n of quinones. However there was an increased - 120 -TABLE XI E f f e c t of s i l i c a g e l column eluates on NADPH o x i d a t i o n F r a c t i o n Eluent number Percent of Incubation 5 c o n t r o l a c t i v i t y time (minutes) 15 1 Hexane 58 77 2 Hexane 114 91 3 Hexane 114 83 4 Hexane 73 82 5 Hexane 82 100 6 Hexane-c h l o r o f orm (19:1) 82 91 7 Hexane-c h l o r o f orm (9:1) 100 97 8 Hexane-c h l o r o f orm (4:1) 100 97 9 Hexane-c h l o r o f orm (1.5:1) 100 100 10 Chloroform 418 448 11 Chloroform 435 425 12 Chloroform 535 489 Incubations were c a r r i e d out as described i n Table X. 0.05 ml of each column el u a t e d i s s o l v e d i n ethanol was added to the r e a c t i o n mixture. - 121 -a b s o r p t i o n a t s h o r t e r w a v e l e n g t h s i n d i c a t i n g t h e p r e s e n c e o f e x t r a n e o u s m a t e r i a l . A l l a t t e m p t s a t f u r t h e r p u r i f i c a t i o n o f t h e e l u a t e s s t i m u l a t i n g NADPH o x i d a t i o n r e s u l t e d i n a l o s s o f t h i s a c t i v i t y . M o r e o v e r , a d d i t i o n o f a u t h e n t i c c o enzyme Qio t o t h e enzyme p r e p a r a t i o n d i d n o t p r o d u c e a n y s t i m u l a t i o n o f NADPH o x i d a t i o n . B e c a u s e o f t h e i n s o l u b i l i t y o f coenzyme Qio i n a q u e o u s m e d i a i t h a s b e e n d i f f i c u l t t o d e m o n s t r a t e t h a t i t f u n c t i o n s i n e l e c t r o n t r a n s p o r t e v e n t h o u g h s u c h a f u n c t i o n h a s b e e n i m p l i e d ( 1 5 1 - 1 5 4 ) . E x p e r i m e n t s w e r e t h e r e f o r e c a r r i e d o u t t o d e t e r m i n e w h e t h e r coenzyme Q i 0 i n -s o l u b i l i t y was r e s p o n s i b l e f o r i t s i n a b i l i t y t o s t i m u l a t e NADPH o x i d a t i o n i n t h e l l g - h y d r o x y l a s e p r e p a r a t i o n . Coenzyme Qio was a d d e d i n t h e p r e s e n c e o f T r i t o n X-100, an a g e n t u s e d t o s o l u b i l i z e q u i n o n e s i n e l e c t r o n t r a n s p o r t s t u d i e s , a n d i n t h e f o r m o f a coenzyme Q - p h o s p h o l i p i d m i c e l l e p r e p a r e d w i t h a s o l e c t i n . N e i t h e r o f t h e s e t r e a t m e n t s r e s u l t e d i n s t i m u -l a t i o n o f NADPH o x i d a t i o n by coenzyme Q i 0 . T h e r e f o r e i t i s c o n c l u d e d t h a t a l t h o u g h coenzyme Q i 0 i s a component o f t h e s i l i c a g e l c o l u m n e l u a t e s t h a t s t i m u l a t e NADPH o x i d a t i o n , i t a l o n e may n o t be r e s p o n s i b l e f o r t h i s s t i m u l a t i o n . O t h e r c o m p o n e n t s o f t h e n e u t r a l l i p i d f r a c t i o n o f m i t o c h o n d r i a c a p a b l e o f r e v e r s i b l e o x i d a t i o n a n d r e d u c t i o n h a v e b e e n i s o -l a t e d ( 1 5 0 ) . The p o s s i b i l i t y e x i s t s t h a t t h e component r e s p o n s i b l e f o r t h i s s t i m u l a t i o n i s m e r e l y e l u a t e d w i t h c oenzyme Qio a n d may be s i m i l a r i n c h e m i c a l a n d p h y s i c a l p r o p e r t i e s t o t h i s q u i n o n e . A l t e r n a t i v e l y coenzyme Q may - 122 -a c t i n c o n j u n c t i o n w i t h some other compound to s t i m u l a t e NADPH o x i d a t i o n . Experiments were c a r r i e d out to determine whether the o x i d a t i o n of NADPH by the enzyme p r e p a r a t i o n e x t r a c t e d from the a d r e n a l m i t o c h o n d r i a l acetone powder was due to the presence of a bound quinone-type compound. Four g of m i t o c h o n d r i a l acetone powder were s a p o n i f i e d w i t h 8 g NaOH, 2 g p y r o g a l l o l , 40 ml methanol and 30 ml of H 20 f o r one hour (113). The r e f l u x e d mixture was e x t r a c t e d with h e x a n e . , the .'hexane f r a c t i o n was con c e n t r a t e d and a p p l i e d t o a m a g n e s o l - c e l i t e (5:1) column. The column was developed as d e s c r i b e d by Bragg and P o l g l a s e (114) . The a b s o r p t i o n s p e c t r a of the e l u a t e f r a c t i o n s gave no i n d i c a t i o n of the presence of v i t a m i n K or coenzyme Q. T h e r e f o r e , w i t h -i n the l i m i t s of the method employed, i t must be concluded t h a t the o x i d a t i o n of NADPH by the enzyme p r e p a r a t i o n i s not mediated by a quinone. (e) S t i m u l a t i o n of l l g - H y d r o x y l a t i o n by the Ace t o n e - E x t r a c t e d  L i p i d F r a c t i o n and by A s o l e c t i n The f r a c t i o n a t i o n s t u d i e s o u t l i n e d i n Table IX suggest t h a t a p h o s p h o l i p i d p r e s e n t i n t h i s acetone e x t r a c t i s r e -s p o n s i b l e f o r the s t i m u l a t i o n of l l g - h y d r o x y l a t i o n . A com-p a r i s o n of the e f f e c t s of the ac e t o n e - e x t r a c t e d l i p i d and a s o l e c t i n , a commercial p l a n t p h o s p h o l i p i d p r e p a r a t i o n , on l l g r - h y d r o x y l a t i o n was t h e r e f o r e c a r r i e d out. - 123 -T h e e f f e c t s o f t h e a c e t o n e - e x t r a c t e d l i p i d f r a c t i o n a n d a s o l e c t i n o n t h e c o u r s e o f l l g - h y d r o x y l a t i o n a r e shown i n F i g u r e 2 8 . A d d i t i o n o f t h e a c e t o n e - e x t r a c t e d l i p i d ( 2 . 4 m g / m l , f i n a l c o n c e n t r a t i o n ) r e s u l t s i n a 1 . 7 - f o l d i n c r e a s e i n t h e i n i t i a l r a t e o f l l g - h y d r o x y l a t i o n . I n a d d i t i o n , t h e r a t e o f h y d r o x y l a t i o n i n t h e p r e s e n c e o f l i p i d r e m a i n s l i n e a r t h r o u g h o u t t h e c o u r s e o f i n c u b a t i o n , w h i l e i n t h e c o n t r o l t h e r a t e d e c r e a s e s i n t h e l a t t e r s t a g e s o f i n c u b a t i o n . T h i s r e -s u l t s u g g e s t s t h a t t h e l i p i d f r a c t i o n i s s t a b i l i z i n g a l a b i l e c o m p o n e n t r e q u i r e d f o r h y d r o x y l a t i o n . T h e s t i m u l a t i o n o f l l g - h y d r o x y l a t i o n by t h e a c e t o n e - e x t r a c t c a n a l s o b e p r o -d u c e d b y a s o l e c t i n . T h i s p h o s p h o l i p i d p r e p a r a t i o n , a t a c o n c e n t r a t i o n e q u i v a l e n t t o t h a t o f t h e a c e t o n e - e x t r a c t e d l i p i d ( 2 . 4 m g / m l ) , p r o d u c e s a 1 . 4 - f o l d i n c r e a s e i n t h e i n i t i a l r a t e o f l l g - h y d r o x y l a t i o n . T h e s t i m u l a t i o n o f l l g - h y d r o x y l a t i o n a s a f u n c t i o n o f l i p i d c o n c e n t r a t i o n i s shown i n F i g u r e 2 9 . A d d i t i o n o f i n c r e a s i n g q u a n t i t i e s o f t h e a c e t o n e - e x t r a c t l i p i d f r a c t i o n r e s u l t s i n a n i n c r e a s e i n l l g - h y d r o x y l a t i o n , a m a x i m a l s t i m u -l a t i o n o f 30% b e i n g a c h i e v e d a t a l i p i d c o n c e n t r a t i o n o f 1 m g / m l . A s o l e c t i n , h o w e v e r , p r o d u c e s 55% s t i m u l a t i o n a t t h e l o w e s t c o n c e n t r a t i o n t e s t e d ( 0 . 2 5 m g / m l ) a n d f u r t h e r i n c r e a s e s i n a s o l e c t i n c o n c e n t r a t i o n c a u s e a d i m i n u t i o n i n i t s s t i m u -l a t o r y e f f e c t . T h i s l o s s o f s t i m u l a t o r y a c t i v i t y a t h i g h e r a s o l e c t i n c o n c e n t r a t i o n s s u g g e s t s t h a t t h e p h o s p h o l i p i d p r e p a r a t i o n c o n t a i n s a c o m p o n e n t t h a t i n h i b i t s l l g - h y d r o x y -l a s e a c t i v i t y a t h i g h e r c o n c e n t r a t i o n s o f t h e p h o s p h o l i p i d p r e p a r a t i o n . 0 8 16 2 4 INCUBATION T IME , min F i g . 2 8 . E f f e c t o f m i t o c h o n d r i a l l i p i d a n d a s o l e c t i n o n t h e H B - h y d r o x y l a t i o n o f d e o x y c o r t i c o s t e r o n e . E a c h r e a c t i o n f l a s k c o n t a i n e d : d e o x y c o r t i c o s t e r o n e - 1 , 2 -3 H ( s p e c i f i c a c t i v i t y 5 . 6 3 x 1 0 6 c p m / u m o l e ) d i s s o l v e d i n 0 . 0 5 m l o f p r o p y l e n e g l y c o l , 54 u m o l e s / 1 , f i n a l c o n c e n t r a t i o n ; 1 .0 m l o f T r i s - M g C l 2 b u f f e r ; 2 . 0 m l o f enzyme p r e p a r a t i o n ; a n d 0 . 2 m l o f l i p i d s u s p e n s i o n c o n t a i n i n g 40 mg l i p i d / m l . A f t e r p r e -i n c u b a t i o n f o r 8 m i n u t e s , NADPH ( 4 . 4 u m p l e s ) d i s s o l v e d i n 0 . 1 m l T r i s - M g C l z b u f f e r was a d d e d . A s a m p l e ( 1 . 0 ml) was r e -moved f r o m e a c h r e a c t i o n f l a s k a f t e r 8 , 1 6 , a n d 24 m i n u t e s o f i n c u b a t i o n a n d p i p e t t e d i n t o e t h y l a c e t a t e (2 ml) t o t e r m i n a t e t h e r e a c t i o n . C o n t r o l ( © ) ; p l u s a c e t o n e - e x t r a c t e d l i p i d (A ) ; p l u s a s o l e c t i n (o). - 125 -160 [LIPID] mg/ml F i g . 29. 113-hydroxylation of deoxycorticosterone as a func-t i o n of m i t o c h o n d r i a l l i p i d or a s o l e c t i n c o n c e n t r a t i o n . Incubations were c a r r i e d out as described i n Table IX. Deoxycorticosterone-1,2- 3H ( s p e c i f i c a c t i v i t y 4.58 x 10 7 cpm/ ymole) d i s s o l v e d i n 0.05 ml of propylene g l y c o l was added to each r e a c t i o n f l a s k to give a f i n a l c o n c e n t r a t i o n of 12.8 ymoles/1. M i t o c h o n d r i a l l i p i d or a s o l e c t i n (0, 0.5, 1.0, 2.0, 4.0 or 8.0 mg) was added suspended i n 0.2 ml of T r i s - M g C l 2 b u f f e r . Acetone-extracted l i p i d (©); a s o l e c t i n (o). - 126 -Cytochrome P-4 50 possesses hydrophobic p r o p e r t i e s due to i t s p h o s p h o l i p i d content or environment (90,93,94). There-f o r e the p o s s i b i l i t y t h a t the phospholipid s t i m u l a t i o n of l l g -h y d r o x y l a t i o n was a r e s u l t of some e f f e c t on cy'-t.ochrome P-450 was deemed worthy of examination. The e f f e c t of the acetone-e x t r a c t e d l i p i d f r a c t i o n on cytochrome P-450 i n both the presence and absence of deoxycorticosterone s u b s t r a t e i s shown i n F i g u r e 30. The cytochrome was measured under experimental c o n d i t i o n s i d e n t i c a l w i t h those employed f o r measurement of l l g - h y d r o x y l a s e a c t i v i t y shown i n Figure 29. A d d i t i o n of deoxycorticosterone (12.7 ymoles/1, f i n a l concentration) i n -creases the recovery of cytochrome P-450 as has been pre-v i o u s l y demonstrated ( F i g . 17). In the presence of the acetone-e x t r a c t e d l i p i d f r a c t i o n an increase i n cytochrome P-450 i s observed which i s greater than t h a t produced by the subs t r a t e deoxycorticosterone. No a d d i t i o n a l increase i n cytochrome P-450 i s noted when deoxycorticosterone and l i p i d are both present i n the r e a c t i o n mixture. Thus the e f f e c t s of deoxy-c o r t i c o s t e r o n e and the acetone-extracted l i p i d on cytochrome P-450 are not a d d i t i v e . The increase i n cytochrome P-4 50 produced by the a c e t o n e - l i p i d f r a c t i o n can e x p l a i n the stimu-l a t i o n of l l g - h y d r o x y l a t i o n by t h i s f r a c t i o n observed i n Figures 2.8, 29 and Table IX. The f a c t o r present i n the acetone-extracted l i p i d f r a c -t i o n producing the s t i m u l a t i o n i n l l g - h y d r o x y l a s e a c t i v i t y i s p r e c i p i t a t e d from acetone at -20° C CTable I X I . The data shown i n Figure 31 r e v e a l that increased cytochrome - 127 -d E O O in i o in UJ O < CD or o CO CO < 0.0 4 0.02 0 J i 1 ! ; j C O N T R O L M I T O -CHONDRIAL LIPID F i g . 30. E f f e c t of deoxycorticosterone and m i t o c h o n d r i a l l i p i d on cytochrome P-4 50. Appropriate r e a c t i o n f l a s k s contained: d e o x y c o r t i c o -sterone (12.7 umoles/1, f i n a l concentration) d i s s o l v e d i n 0.15 ml of propylene g l y c o l ; 1.65 ml of T r i s - M g C l 2 b u f f e r , 3.90 ml of enzyme p r e p a r a t i o n ; and 0.6 ml of a l i p i d sus-pension (10 mg l i p i d / m l ) . A f t e r 8 minutes p r e - i n c u b a t i o n , NADPH (3 umoles) was added d i s s o l v e d i n 0.15 ml of T r i s - M g C l 2 ' b u f f e r to each r e a c t i o n f l a s k . S o l i d ammonium s u l f a t e to giv e 20% s a t u r a t i o n was then added a f t e r 90 seconds of i n -cubation and cytochrome P-450 was i s o l a t e d and measured as desc r i b e d i n the methods. Without d e o x y c o r t i c o s t e r o n e , c l e a r bars; plus deoxy-c o r t i c o s t e r o n e , shaded bars. - 1 2 8 CONTROL PLUS P L U S TOTAL ACETONE MITO- (-20°C) C H O N D R I A L INSOLUBLE L I P ID FRACTION F i g . 31.^ E f f e c t of t o t a l m i t o c h o n d r i a l l i p i d and -20° C ac e t o n e - i n s o l u b l e f r a c t i o n on cytochromes P-450 and P-420. Incubations were c a r r i e d out as described i n F i g . 30. 0.15 ml of propylene g l y c o l was added to each r e a c t i o n f l a s k i n s t e a d of deo x y c o r t i c o s t e r o n e . The l i p i d f r a c t i o n s (6 mg) were added suspended i n T r i s - M g C l 2 b u f f e r (0.6 ml). Cytochrome P-450, c l e a r bars; cytochrome P-420, shaded bars. - 129 -P-450 l e v e l s c a n a l s o be p r o d u c e d b y t h i s p r e c i p i t a t e d f a c t o r , a l t h o u g h t o a s l i g h t l y l e s s e r d e g r e e t h a n t h a t p r o d u c e d w i t h t h e t o t a l l i p i d f r a c t i o n . T h i s w o u l d i n d i c a t e t h a t t h e p h o s -p h o l i p i d p r e s e n t i n t h e l i p i d f r a c t i o n i s r e s p o n s i b l e f o r t h e e l e v a t i o n i n c y t o c h r o m e P-4 50 c o n t e n t . F i g u r e 32 shows t h a t t h e i n c r e a s e i n c y t o c h r o m e P-450 r e c o v e r e d a f t e r t h e i n c u b a t i o n p e r i o d i s d e p e n d e n t u p o n t h e c o n c e n t r a t i o n o f t h e a c e t o n e - e x t r a c t e d l i p i d f r a c t i o n p r e s e n t i n t h e r e a c t i o n m i x t u r e . The e f f e c t .of i n c r e a s i n g c o n c e n -t r a t i o n s o f l i p i d on c y t o c h r o m e P-450 ( F i g . 32) i s s i m i l a r t o t h e e f f e c t o f l i p i d on l l g - h y d r o x y l a t i o n ( F i g . 2 9 ) , b o t h e f f e c t s b e i n g m a x i m a l a t a l i p i d c o n c e n t r a t i o n o f a p p r o x i -m a t e l y 1 mg/ml. The s i m i l a r i t y o f t h e two c u r v e s s u g g e s t s t h a t t h e o b s e r v e d s t i m u l a t i o n o f l l g - h y d r o x y l a t i o n r e s u l t s f r o m i n c r e a s e d c y t o c h r o m e P-450. C y t o c h r o m e P-420 i s a l s o i n c r e a s e d i n t h e p r e s e n c e o f t h e l i p i d e x t r a c t ( F i g . 32) a l t h o u g h t o a l e s s e r e x t e n t . A s o l e c t i n p r o d u c e s i n c r e a s e s i n c y t o c h r o m e P-4 50 l e v e l s a t c o n c e n t r a t i o n s l o w e r t h a n t h e l i p i d e x t r a c t , a t w o - f o l d i n c r e a s e i n c y t o c h r o m e P-450 b e i n g o b s e r v e d i n t h e p r e s e n c e o f a s o l e c t i n a t a c o n c e n t r a t i o n o f 0.23 mg/ml ( F i g . 3 3 ) . As t h e a s o l e c t i n c o n c e n t r a t i o n i n c r e a s e s , c y t o -c hrome P-450 d i m i n i s h e s . A s i m i l a r e f f e c t o f a s o l e c t i n i s s e e n on c y t o c h r o m e P-420. The d e c r e a s e i n c y t o c h r o m e P-450 a t h i g h e r a s o l e c t i n c o n c e n t r a t i o n s may be due t o l y s o l e c i -t h i n i n t h e p h o s p h o l i p i d p r e p a r a t i o n . L y s o l e c i t h i n h a s d e t e r g e n t a c t i o n a n d h a s b e e n shown t o c a u s e t h e d e c o m p o s i t i o n - 130 -0.05 o 0.0 1 -m • o 1 1 1 1 ' 0 • 1.0 2.0 [LIPID EXTRACT ] , mg/ml F i g . 32. Recovery of cytochromes P-450 and P-420 as a func-t i o n of the m i t o c h o n d r i a l l i p i d c o n c e n t r a t i o n . Each r e a c t i o n f l a s k contained: 0.15 ml of propylene g l y c o l ; 1.05 ml of T r i s - M g C l 2 b u f f e r ; 3.90 ml of enzyme pre-p a r a t i o n ; and 0.0, 0.15, 0.30, 0.60, or 1.2 ml of the mito-c h o n d r i a l l i p i d suspension (10 mg l i p i d / m l ) . The volume of each r e a c t i o n mixture was adjusted to 6.3 ml w i t h T r i s - M g C l 2 b u f f e r . A f t e r 8 minutes p r e - i n c u b a t i o n , NADPH (3 umoles) was added d i s s o l v e d i n 0.15 ml of T r i s - M g C l 2 b u f f e r to each r e a c t i o n f l a s k . S o l i d ammonium s u l f a t e was then added to give 20% s a t u r a t i o n a f t e r 90 seconds of i n c u b a t i o n and cyto-chromes P-450 and P-420 were i s o l a t e d and measured as described i n the methods. Cytochrome P-450 (o); cytochrome P-420 (©). - 131 -[ASOLECT IN ] , m g / m l F i g . 33. Recovery of cytochromes P-450 and P-420 as a f u n c t i o n of a s o l e c t i n c o n c e n t r a t i o n . Incubations were c a r r i e d out as described i n F i g . 32. 0.0, 0.08, 0.15, or 0.6 ml of the a s o l e c t i n suspension (10 mg a s o l e c t i n / m l ) was added to the r e a c t i o n mixture. Cytochrome P-450 (o); cytochrome P-420 (©). - 132 -of cytochrome P-450 to P-420. The decrease i n cytochrome P-450 produced by higher concentrations of a s o l e c t i n i s very s i m i l a r to the decrease i n l l g - h y d r o x y l a s e s t i m u l a t i o n observed at increased a s o l e c t i n concentrations ( F i g . 29) and i n d i c a t e s a r e l a t i o n s h i p between these two phenomena. In order to e s t a b l i s h the mechanism whereby the acetone-e x t r a c t e d l i p i d f r a c t i o n was producing an increased recovery of cytochrome P-450 ( F i g . 32), the rat e of breakdown of cyto-chrome P-450 was measured i n the presence and absence of the acetone-extracted l i p i d f r a c t i o n . The r e s u l t s are i l l u s t r a t e d i n F igure 34. Incubation of the enzyme pr e p a r a t i o n at 37° C r e s u l t s i n a decrease i n recoverable cytochrome P-4 50 and a corresponding increase i n cytochrome P-420, as has been pre-v i o u s l y observed ( F i g . 16). In the presence of the acetone-e x t r a c t e d l i p i d f r a c t i o n (0.93 mg/ml) there i s an increase i n cytochrome P-4 50 recovery when compared to the c o n t r o l . The increase i n cytochrome P-450 i n the zero-time c o n t r o l (main-tained at 4° C) i n the presence of the acetone-extracted l i p i d i s an unexpected f i n d i n g . Previous evidence has i n d i -cated t h a t cytochrome P-450 i s r e l a t i v e l y s t a b l e at lower temperatures ( F i g . 15) and very l i t t l e decomposition of the hemoprotein would be expected at 4° C. The d i f f e r e n c e i n cytochrome P-450 l e v e l s at zero time may i n d i c a t e that the acetone-extracted l i p i d f r a c t i o n i s enabling r e c o n s t i t u t i o n of cytochrome P-450 from cytochrome P-420. The reconversion of microsomal cytochrome P-420 to cytochrome P-4 50 has been reported by Ichikawa and Yamano (94). The reconversion of - 133 -d. 0 .08 E O o INCUBATION TIME , min F i g . 34. E f f e c t of m i t o c h o n d r i a l l i p i d on the r a t e of cytochrome P-450 disappearance. Appropriate r e a c t i o n f l a s k s contained: 0.15 ml of propylene g l y c o l ; 1.65 ml of T r i s - M g C l 2 b u f f e r ; 3.90 ml of enzyme p r e p a r a t i o n and 0.6 ml of a m i t o c h o n d r i a l l i p i d suspension (10 mg l i p i d / m l ) . S o l i d ammonium s u l f a t e was added to gi v e 20% s a t u r a t i o n a f t e r 0, 4, 8 and 12 minutes of i n c u b a t i o n and cytochromes P-450 and P-420 were i s o l a t e d and measured as described i n the methods. Cytochrome P-450: c o n t r o l (© ©) ; plus m i t o c h o n d r i a l l i p i d (o o) . Cytochrome P-420: c o n t r o l (•© © ) ; plus mito-c h o n d r i a l l i p i d (o o ) . - 134 -detergent-produced cytochrome P-420 to cytochrome P-450 was produced by p o l y o l s such as g l y c e r o l or ethylene g l y c o l , and at temperatures as low as 0° C. The p o s s i b i l i t y t h a t the acetone-extracted l i p i d f r a c t i o n i s achieving a s i m i l a r r e -conversion of cytochrome P-420 to cytochrome P-450 i n the enzyme system from an adrenal m i t o c h o n d r i a l acetone powder cannot be disregarded. Incubation of the enzyme pr e p a r a t i o n i n the presence of acetone-extracted l i p i d , a t 37° C, r e s u l t s i n an i n i t i a l r a p i d decrease i n cytochrome P-450 to the l e v e l of the c o n t r o l i n c u b a t i o n at zero time ( F i g . 34). Thus the r e c o n s t i t u t e d cytochrome P-4 50 formed i n the presence of l i p i d must be extremely l a b i l e and undergo spontaneous de-composition at elevated temperatures. Upon f u r t h e r incuba-t i o n however, there i s no a d d i t i o n a l l o s s of cytochrome P-450 i n the presence of the acetone-extracted l i p i d ( F i g . 34). Thus t h i s l i p i d f r a c t i o n must a l s o s t a b i l i z e cytochrome P-450. DISCUSSION The i n h i b i t i o n of 116-hydroxylation by dicumarol suggested the involvement of a quinone-type compound i n the hydroxyla-t i o n r e a c t i o n , s i n c e dicumarol i s known to e f f e c t a number of p y r i d i n e nucleotide-quinone reductases (135-141). The enzyme pr e p a r a t i o n e x t r a c t e d from an acetone powder of adrenal mitochondria does indeed e x h i b i t a naphthoquinone-stimulated o x i d a t i o n of NADPH. This NADPH-quinone reductase a c t i v i t y has p r o p e r t i e s s i m i l a r to those of microsomal NADPH-cytochrome c reductase (62). - 135 -B o t h m e n a d i o n e ( 2 - m e t h y l - l , ^ n a p h t h o q u i n o n e ) a n d 1,4-n a p h t h o q u i n o n e s t i m u l a t e t h e a e r o b i c o x i d a t i o n o f NADPH by t h e m i t o c h o n d r i a l enzyme s y s t e m , b u t 1 , 4 - t o l u o q u i n o n e a n d 1,4-b e n z o q u i n o n e a r e r e l a t i v e l y i n e f f e c t i v e . S i m i l a r s p e c i f i -c i t y h a s b e e n r e p o r t e d i n o t h e r q u i n o n e - d e p e n d e n t NADPH o x i -d a s e s (136,137,155). H o w e v e r , d i c u m a r o l s t i m u l a t e s t h e q u i n o n e -d e p e n d e n t a c t i v i t y i n t h e a d r e n a l m i t o c h o n d r i a l a c e t o n e powder p r e p a r a t i o n . D i c u m a r o l must be r e l e a s i n g o r a f f e c t i n g some f a c t o r i n v o l v e d i n t h i s o x i d a t i o n r e a c t i o n w h i c h r e s u l t s i n an e x t e n s i v e i n c r e a s e i n NADPH o x i d a t i o n i n t h e p r e s e n c e o f n a p h t h o q u i n o n e s . I n c o n t r a s t , t h e q u i n o n e r e d u c t a s e o f t h e m i c r o s o m a l a n d s o l u b l e f r a c t i o n s o f l i v e r h a v e b e e n r e p o r t e d t o be e i t h e r i n h i b i t e d b y (136,137) o r i n s e n s i t i v e t o (146) d i c u m a r o l . A l t h o u g h n a p h t h o q u i n o n e s s t i m u l a t e t h e o x i d a t i o n o f NADPH, t h e y do- n o t ha v e t h i s e f f e c t on l l g - h y d r o x y l a t i o n . I n d e e d , u n d e r c o n d i t i o n s w h e r e NADPH i s r a t e - l i m i t i n g , mena-d i o n e c a n a c t u a l l y p r o d u c e a n i n h i b i t i o n o f l l g - h y d r o x y l a t i o n . T h u s , t h e r e must be a c o m p e t i t i o n b e t w e e n m e n a d i o n e and t h e l l g - h y d r o x y l a s e f o r NADPH. A s i t e f o r t h e i n t e r a c t i o n o f m e n a d i o n e w i t h t h e l l g - h y d r o x y l a s e p a t h w a y was f i r s t s u g g e s t e d by K i m u r a (60) when he o b s e r v e d t h a t a d r e n o d o x i n r e d u c t a s e , a c o n s t i t u e n t o f t h e l l g - h y d r o x y l a s e p a t h w a y , c o u l d u t i l i z e e l e c t r o n a c c e p t o r s s u c h a s c y t o c h r o m e c, m e n a d i o n e , a n d f e r r i -c y a n i d e i n p l a c e o f t h e n a t u r a l a c c e p t o r , a d r e n o d o x i n . S i m i l a r o b s e r v a t i o n s h a v e b e e n made i n p r e s e n t s t u d i e s . M o r e o v e r , c y t o c h r o m e P-420 c a n i n t u r n a c c e p t e l e c t r o n s f r o m m e n a d i o n e , - 136 -or menadione can f u n c t i o n as a c o f a c t o r i n a NADPH-cytochrome P-420 reductase present i n t h i s enzyme system. A summary of the i n t e r a c t i o n of menadione w i t h the 113-hydroxylase pathway may be o u t l i n e d as shown i n Figure 35. El e c t r o n s can be t r a n s -f e r r e d from NADPH v i a adrenodoxin reductase and menadione to cytochrome P-420, which can i n t e r a c t w i t h oxygen to r e -generate the o x i d i z e d form of the cytochrome (89). Menadione i s a l s o a u t o x i d i z a b l e (146,147). Thus the p o s s i b i l i t y t h a t the o x i d i z e d form of t h i s quinone i s regenerated by i n t e r -a c t i o n w i t h molecular oxygen as w e l l as w i t h cytochrome P-420 cannot be disregarded. The scheme presented here ( F i g . 35) i l l u s t r a t e s the p o s s i b l e c a t a l y t i c r o l e of menadione i n NADPH o x i d a t i o n , e x p l a i n i n g i t s a b i l i t y to r a p i d l y o x i d i z e q u a n t i -t i e s of NADPH (0.6 ymole)' i n excess of that expected f o r the red u c t i o n of the added menadione (0.05 ymole) ( F i g . 22). The lack of e f f e c t of menadione on 113-hydroxylation i n the presence of excess NADPH i n d i c a t e s i t s non-involvement i n t h i s r e a c t i o n sequence and suggests that dicumarol i n h i -b i t i o n of the 113-hydroxylase i s not mediated by i t s e f f e c t on t h i s type of r e a c t i o n . Indeed, a d d i t i o n of dicumarol to the i n c u b a t i o n mixture i n the presence of menadione r e s u l t s i n an increased i n h i b i t i o n of 113-hydroxylation due to the extremely r a p i d o x i d a t i o n of NADPH i n the presence of both dicumarol and menadione. Kimura a l s o observed (6 0) t h a t adrenodoxin reductase may u t i l i z e cytochrome a as an e l e c t r o n acceptor i n s t e a d of adreno-doxin. Omura et al. (57) reported that p u r i f i e d adrenodoxin NADPH Adrenodoxin Cytochrome -> Reductase > Adrenodoxin >- P-450 Menadione Cytochrome + P-420 Deoxycorticosterone 0; Co r t i c o s t e r o n e + H 20 0 2 0 2 F i g . 35. Schematic r e p r e s e n t a t i o n of the i n t e r a c t i o n of menadione with the components of the e l e c t r o n t r a n s p o r t pathway f o r 113-hydroxylation. - 1 3 8 -r e d u c t a s e f a i l e d t o c a t a l y z e t h e r e d u c t i o n o f c y t o c h r o m e o by NADPH u n l e s s a d r e n o d o x i n was a l s o p r e s e n t . S p e c t r o p h o t o -m e t r i c e v i d e n c e i n d i c a t e s t h e p r e s e n c e o f NADPH-cytochrome o r e d u c t a s e a c t i v i t y i n t h e enzyme p r e p a r a t i o n e m p l o y e d i n t h e p r e s e n t s t u d i e s ( F i g . 7 ) . T h i s r e d u c t i o n o f c y t o c h r o m e c i s l i k e l y m e d i a t e d by t h e a d r e n o d o x i n r e d u c t a s e d e s c r i b e d by K i m u r a (60) a n d Omura et al. ( 5 7 ) . The s t i m u l a t i o n o f NADPH o x i d a t i o n by t h e a c e t o n e - e x t r a c t e d l i p i d f r a c t i o n i s v e r y s i m i l a r t o t h a t o b s e r v e d w i t h n a p h t h o -q u i n o n e s ( F i g s . 22 and 2 3 ) . B o t h s t i m u l a t o r y p r o c e s s e s a r e a f f e c t e d by d i c u m a r o l i n a n a n a l o g o u s manner ( T a b l e V I I ) . H o w e v e r , w h e t h e r t h e s t i m u l a t i o n o f NADPH o x i d a t i o n b y t h e a c e t o n e - e x t r a c t e d l i p i d f r a c t i o n i s due t o t h e p r e s e n c e o f a q u i n o n e c a n n o t be s t a t e d w i t h c e r t a i n t y . A t t e m p t s t o i s o l a t e t h e s t i m u l a t o r y f a c t o r f r o m t h e l i p i d e x t r a c t w e r e i n c o n c l u -s i v e . A l l a c t i v i t y was f o u n d t o r e s i d e i n f r a c t i o n s c o n t a i n -i n g coenzyme Q 1 0 b u t f u r t h e r a t t e m p t s a t p u r i f i c a t i o n r e -s u l t e d i n a l o s s o f s t i m u l a t o r y a c t i v i t y . F u r t h e r m o r e , a d d i -t i o n o f a u t h e n t i c coenzyme Ch 0 t o t h e enzyme p r e p a r a t i o n u n d e r a v a r i e t y o f c o n d i t i o n s e m p l o y e d t o s o l u b i l i z e t h e q u i n o n e d i d n o t r e s u l t i n any s i g n i f i c a n t s t i m u l a t i o n o f NADPH o x i d a t i o n . S o t t o c a s a a n d C r a n e (150) h a v e i s o l a t e d f o u r c o m p o n e n t s o f t h e n e u t r a l l i p i d f r a c t i o n o f b e e f h e a r t m i t o c h o n d r i a c a p a b l e o f u n d e r g o i n g r e v e r s i b l e o x i d a t i o n a n d r e d u c t i o n . One o f t h e s e c o m p o n e n t s was f o u n d t o be c o -enzyme Q i o / a n o t h e r was f o u n d i n t h e f r a c t i o n c o n t a i n i n g a -t o c o p h e r o l , w h i l e t h e n a t u r e o f t h e o t h e r two c o m p o n e n t s c o u l d - 139 -not be c l e a r l y e s t a b l i s h e d . Sottocasa and Crane observed that one unknown component behaved chromatographically s i m i -l a r ' to known members of the v i t a m i n K f a m i l y , w h i l e the other had s p e c t r a l p r o p e r t i'es suggesting a benzoquinone type of s t r u c t u r e . Of i n t e r e s t i s the statement of Sottocasa and Crane t h a t t h i s component e x h i b i t e d a spectrum s i m i l a r to that of coenzyme Q 1 0 and was not e a s i l y separated from co-enzyme Qio on s i l i c i c a c i d column chromatography. I t i s p o s s i b l e t h a t a s i m i l a r component i s present i n the l i p i d e x t r a c t of adrenal mitochondria and that i t i s r e s p o n s i b l e f o r the observed s t i m u l a t i o n of NADPH o x i d a t i o n . The s t i m u l a t i o n of l l g - h y d r o x y l a t i o n by the acetone-e x t r a c t e d l i p i d f r a c t i o n may be a t t r i b u t e d to the phospho-l i p i d components of the l i p i d e x t r a c t . The s t i m u l a t o r y f a c t o r can be p r e c i p i t a t e d from acetone at -20° C and i t s a c t i v i t y can be p a r t i a l l y mimicked by a s o l e c t i n , a soybean p h o s p h o l i p i d p r e p a r a t i o n . The s t i m u l a t o r y e f f e c t of t h i s p h o s p h o l i p i d component of the acetone-extracted l i p i d may be due to s t a b i l i z a t i o n of cytochrome P-450. Moreover, the pho s p h o l i p i d may produce a r e c o n s t i t u t i o n of cytochrome P-450 from cytochrome P-4 20 although the r e c o n s t i t u t e d hemoprotein appears to be extremely l a b i l e . Thus the acetone-extracted l i p i d f r a c t i o n contains the l i p i d moiety of cytochrome P-450 removed during p r e p a r a t i o n of the acetone powder; and r e -combination of t h i s moiety w i t h cytochrome P-420 produces the cytochrome P-450 complex. P o l y o l s such as g l y c e r o l can pro-t e c t cytochrome P-450 from decomposition (80,94) and e f f e c t - 140 -a r e c o n s t i t u t i o n of t h i s hemoprotein from cytochrome P-4 20 (94). The e f f e c t s of p o l y o l s are analogous to the phospho-l i p i d e f f e c t on cytochrome P-450 demonstrated i n the present experiments. The mechanism whereby phospholipid s t a b i l i z e s cytochrome P-450 i s unclear. This hemoprotein contains phospholipid and req u i r e s phospholipid to maintain i t s unique p r o p e r t i e s . The importance of l i p i d i n the f u n c t i o n of the mitochondrion has been the subject of much research (110,152,156). Green and Tzagoloff (156) have po s t u l a t e d that p h o s p h o l i p i d m i c e l l e s can i n t e r a c t with b a s i c p r o t e i n s such as cytochrome c to form s t a b l e complexes. Cytochrome P-4 50 may w e l l be such a com-plex and the presence of a d d i t i o n a l p h o s p h o l i p i d may a i d i n s t a b i l i z i n g the cytochrome P-450 complex as w e l l as recon-s t i t u t i n g cytochrome P-450 from cytochrome P-420 produced by removal of phospholipid by acetone. The requirement of c e r t a i n enzymes lo c a t e d i n the inner membrane of the mito-chondrion f o r phospholipid has been e s t a b l i s h e d (156). Examples are the phospholipid requirement of the e l e c t r o n t r a n s p o r t chain (152), g-hydroxybutyrate dehydrogenase (157, 158), and c y t i d i n e diphosphocholine t r a n f e r a s e (156,159). The p a r t i c u l a t e nature of m i t o c h o n d r i a l cytochrome P-4 50 and the i n t e r a c t i o n of the l l g - h y d r o x y l a s e pathway w i t h the c l a s s i c a l electron, t r a n s p o r t chain suggest that t h i s hemo-p r o t e i n may a l s o be lo c a t e d i n the inner membrane of the mitochondrion and thus may e x h i b i t a s i m i l a r p h o s p h o l i p i d requirement. - 141 -The i n a b i l i t y to demonstrate that a quinone-dependent NADPH reductase f u n c t i o n s i n 116-hydroxylation suggests t h a t dicumarol i n h i b i t i o n of 116-hydroxylation does not proceed v i a i t s e f f e c t on a quinone reductase r e a c t i o n f u n c t i o n i n g i n 116-hydroxylation. While a phospholipid component of the l i p i d e x t r a c t of adrenal mitochondria can s t i m u l a t e 116-h y d r o x y l a t i o n , t h i s component does not s i g n i f i c a n t l y over-come the i n h i b i t i o n produced by dicumarol under these e x p e r i -mental c o n d i t i o n s ( F i g . 26). Therefore dicumarol i n h i b i t i o n of 116-hydroxylation must be achieved by a mechanism not yet as c r i b e d to t h i s compound. - 142 -PART I I I DICUMAROL AND METOPIRONE INHIBITION OF 113-HYDROXYLATION Evidence presented i n Part I I of t h i s t h e s i s excluded the p o s s i b i l i t y t h a t the i n h i b i t i o n of l l g - h y d r o x y l a t i o n by d i c u -marol was produced by an i n h i b i t o r y a c t i o n of t h i s compound on a NADPH-quinone reductase. Hence a d e t a i l e d k i n e t i c examina-t i o n of dicumarol i n h i b i t i o n was undertaken i n an attempt to determine i t s mode of a c t i o n . S i m i l a r studies on the mechanism of i n h i b i t i o n of Metopirone, a w e l l e s t a b l i s h e d i n h i b i t o r of l l g - h y d r o x y l a t i o n (42-49), were a l s o c a r r i e d out. I t was hoped t h a t a comparative study of the two i n h i b i t o r s would lead to an understanding of t h e i r modes of a c t i o n and of the general mechanism of l l g - h y d r o x y l a t i o n . RESULTS (a) I n h i b i t i o n of l l g - h y d r o x y l a t i o n by Dicumarol Figure 36a shows the rat e s of o x i d a t i o n of NADPH by the enzyme pr e p a r a t i o n alone, and i n the presence of d e o x y c o r t i -costerone, dicumarol, and deoxycorticosterone plus dicumarol. A d d i t i o n of deoxycorticosterone, to the r e a c t i o n mixture i n -creases the r a t e of NADPH o x i d a t i o n over that of the c o n t r o l . The a d d i t i o n of dicumarol to the enzyme pre p a r a t i o n a l s o causes an increase i n the r a t e of NADPH o x i d a t i o n . However, no f u r t h e r enhancement of NADPH o x i d a t i o n i s observed i n the presence of dicumarol plus deoxycorticosterone. These f i n d -ings i n d i c a t e that dicumarol i n h i b i t s the o x i d a t i o n of that p r o t i o n of the reduced coenzyme p a r t i c i p a t i n g i n s t e r o i d l l g -- 143 -0 1 0 2 0 0 8 1 6 2 4 0 , INCUBATION TIME, min F i g . 36a F i g . 36b F i g . 36a. E f f e c t of dicumarol and deoxycorticosterone on NADPH o x i d a t i o n . Appropriate r e a c t i o n f l a s k s contained: 0.6 ymole of deoxy-c o r t i c o s t e r o n e d i s s o l v e d i n 0.05 ml of propylene g l y c o l ; 1.0 ml of T r i s - M g C l 2 b u f f e r ; 2.0 ml of enzyme p r e p a r a t i o n ; and 0.5 ymole of dicumarol d i s s o l v e d i n 0.05 ml of a d i l u t e a l c o h o l i c KOH s o l u t i o n . NADPH (0.8 ymole) i n 0.1 ml of T r i s - M g C l 2 b u f f e r was added at zero time. C o n t r o l (©); plus deoxycorticosterone (o); plus dicumarol (o); plus d e o x y c o r t i c o s t e r o n e and dicumarol (®). F i g . 36b. E f f e c t of dicumarol on the l l g - h y d r o x y l a t i o n of deox y c o r t i c o s t e r o n e . Incubations were c a r r i e d out as described i n F i g . 36a. 0.65 ymole of deoxycorticosterone-1,2- 3H ( s p e c i f i c a c t i v i t y 1.07-x 10 6 cpm/ymole) d i s s o l v e d i n 0.05 ml of propylene g l y c o l was added to each r e a c t i o n f l a s k . A f t e r 8 minutes p r e i n c u -b a t i o n , NADPH (2.2 ymoles) d i s s o l v e d i n C l ml of T r i s - M g C l 2 b u f f e r was added. A sample (1.0 ml) was removed from each f l a s k a f t e r 8, 16, and 24 minutes i n c u b a t i o n , and p i p e t t e d i n t o e t h y l acetate (2 ml) to terminate the r e a c t i o n . C o n t r o l (o); plus dicumarol (@). - 144 -h y d r o x y l a t i o n . The i n h i b i t o r y a c t i o n i s confirmed by the data shown i n Figure 36b. The formation of c o r t i c o s t e r o n e i s great--4 l y reduced by the a d d i t i o n of dicumarol (1.5 x 10 M), being 37% t h a t of the c o n t r o l a f t e r 8 minutes of i n c u b a t i o n . The c o n c e n t r a t i o n range over which dicumarol e x h i b i t s an i n h i b i t o r y a c t i o n on 113-hydroxylation i s shown i n Figure 37. I n h i b i t i o n by t h i s compound i s demonstrable at concentrations of dicumarol as low as 11.6 mumoles/1. At the lower dicumarol concentrations 113-hydroxylation i s r e l a t i v e l y i n s e n s i t i v e to the i n h i b i t o r , a slow l i n e a r increase i n i n h i b i t i o n r e s u l t i n g from a thousandfold increase i n dicumarol c o n c e n t r a t i o n . At the higher dicumarol concentrations there i s a pronounced i n -crease i n the s e n s i t i v i t y of 113-hydroxylation to the i n h i -b i t o r c o n c e n t r a t i o n . The data suggest that dicumarol exerts more than one mode of i n h i b i t i o n of the 113-hydroxylase system. K i n e t i c experiments on the e f f e c t of dicumarol on the i n i t i a l v e l o c i t y of 113-hydroxylation were c a r r i e d out. The e f f e c t of d i f f e r e n t , dicumarol concentrations (58, 145 and 233 umoles/1) on the i n i t i a l r a t e of 113-hydroxylation a t i n c r e a s i n g s u b s t r a t e concentrations i s k i n e t i c a l l y rep-resented i n the p l o t of [S]/v vs[S] i n Figure 38. At the lowest dicumarol c o n c e n t r a t i o n examined (58 umoles/1) d i -cumarol i s a noncompetitive i n h i b i t o r of 113-hydroxylation. However, increases i n i n h i b i t o r c o n c e n t r a t i o n a l t e r the i n h i b i t i o n by dicumarol to that of a mixed type, again sug-g e s t i n g more than one e f f e c t of dicumarol on 113-hydroxylation, - 1 4 5 -i r 100 80 eo o CO X 2 4 0 o UJ a. 20 01 1 1 1 _i —I 0 1 2 3 4 5 6 LOG [DICUMAROL C0NC,mumoles/l] F i g . 37. I n h i b i t i o n of the 116-hydroxylation of d e o x y c o r t i -costerone as a f u n c t i o n of dicumarol c o n c e n t r a t i o n . Each r e a c t i o n f l a s k contained: 0.094 umole of deoxycor-t i c o s t e r o n e - ! , 2- 3H ( s p e c i f i c a c t i v i t y 1.05 x 10 7 cpm/ymole) d i s s o l v e d i n 0.05 ml of propylene g l y c o l ; 1.35 ml of T r i s -MgCl 2 b u f f e r ; 2.6 ml of enzyme p r e p a r a t i o n ; dicumarol, to giv e the i n d i c a t e d f i n a l c o n c e n t r a t i o n , d i s s o l v e d i n 0.1 ml of d i l u t e a l c o h o l i c KOH s o l u t i o n . A f t e r p r e - i n c u b a t i o n f o r 8 minutes, NADPH (3.3 umoles) d i s s o l v e d i n 0.2 ml of T r i s - M g C l 2 b u f f e r was added. A f t e r 20 minutes of i n c u b a t i o n , r e a c t i o n s were terminated by the a d d i t i o n of e t h y l acetate (5 ml) to each f l a s k . - 146 -[S] ( ymoles/1) F i g . 38. [S]/v versus [S] p l o t of dicumarol i n h i b i t i o n of the 116-hydroxylation of d e o x y c o r t i c o s t e r o n e . Each r e a c t i o n f l a s k contained: deoxycorticosterone-1,2-3H ( s p e c i f i c a c t i v i t y , 6.24 x 10 7 cpm/ymole) d i s s o l v e d i n 0.05 ml of propylene g l y c o l ; 0.7 ml of T r i s - M g C l 2 b u f f e r ; 1.3 ml of enzyme p r e p a r a t i o n ; 0, 0.125, 0.313, or 0.5 ymole of dicumarol d i s s o l v e d i n 0.05 ml of a d i l u t e a l c o h o l i c KOH s o l u t i o n . A f t e r 8 minutes of p r e - i n c u b a t i o n , NADPH (1.7 ymoles) d i s s o l v e d i n 0.05 ml of T r i s - M g C l 2 b u f f e r was added. A sample (1.0 ml) was removed from each r e a c t i o n f l a s k a f t e r 90 seconds of incuba-t i o n and p i p e t t e d i n t o e t h y l acetate (2 ml) to terminate the r e a c t i o n . F i n a l c o n c e n t r a t i o n (ymoles/1) of dicumarol: 0 (©), 58 (O) , 145 (Q) , 233 (a) . - 147 -the secondary e f f e c t being observed only at higher dicumarol concentrations. Also worthy of note i s the f a c t t h a t at high dicumarol c o n c e n t r a t i o n s , the experimental values determined cannot be as uniformly j o i n e d by a s t r a i g h t l i n e as at the lowest dicumarol concentration or i n i t s absence. I t i s p o s s i b l e t h a t at high dicumarol concentrations the i n h i b i -t o r y e f f e c t i s complex i n nature and may i n v o l v e s e v e r a l d i f f e r e n t i n t e r a c t i o n s of dicumarol w i t h the 113-hydroxylase system. That more than one i n h i b i t o r y a c t i o n of dicumarol on 113-hydroxylation i s p o s s i b l e i s a l s o demonstrated i n the Dixon p l o t (160) of 1/v vs i shown i n Figure 39. The e f f e c t of i n -cr e a s i n g i n h i b i t o r concentrations at two l e v e l s of sub s t r a t e (7 and 35 umoles/1) i s i l l u s t r a t e d . At dicumarol concentra-t i o n s of 8 0 umoles/1 and lower, the 113-hydroxylase a c t i v i t y i s r e l a t i v e l y i n s e n s i t i v e to changes i n i n h i b i t o r concentra-t i o n as judged by the gradients of the l i n e s j o i n i n g the experimental values. Extension of the l i n e s obtained i n t h i s r e gion of dicumarol c o n c e n t r a t i o n to t h e i r p o i n t of i n t e r -s e c t i o n can e s t a b l i s h both the mode of i n h i b i t i o n and the K. f o r the i n h i b i t o r i However the almost h o r i z o n t a l slopes of the l i n e s obtained at both substrate concentrations prevent an accurate'determination of these parameters. Although one cannot conclude d e f i n i t e l y from the Dixon p l o t ( F i g . 39) that dicumarol at low concentrations i s a noncompetitive i n h i b i t o r of 113-hydroxylation, the k i n e t i c data i n Figure 38 i n d i c a t e noncompetitive i n h i b i t i o n . - 1 4 8 --80 DICUMAROL CONCENTRAT ION (,umoles/l) F i g . 39. 1/v versus i p l o t of dicumarol i n h i b i t i o n of 113-h y d r o x y l a t i o n of deo x y c o r t i c o s t e r o n e . Incubations were c a r r i e d out as described i n F i g . 38. The s p e c i f i c a c t i v i t y of the deoxycorticosterone-1,2- 3H sub-s t r a t e was 4.04 x 10 cpm/ymole. F i n a l c o n c e n t r a t i o n (ymoles/1) of de o x y c o r t i c o s t e r o n e -1,2-3H: 7 ( A ) , 35 ( A ) . - 149 -The secondary e f f e c t of dicumarol on 116-hydroxylation i s evident i n the Dixon p l o t ( F i g . 39) where a d i s t i n c t increase i n the slope of the curve i s apparent as the dicumarol con-c e n t r a t i o n i n c r e a s e s . At the higher deoxycorticosterone c o n c e n t r a t i o n , the p o i n t at which t h i s slope change appears i s at a s l i g h t l y higher dicumarol c o n c e n t r a t i o n and the ex-ten t of the slope change i s decreased. Thus the second d i -cumarol i n h i b i t o r y e f f e c t i s extremely s e n s i t i v e to sub-s t r a t e c o n c e n t r a t i o n , being g r e a t l y diminished at a substrate c o n c e n t r a t i o n of 35 umoles/1. This suggests t h a t the second mode of dicumarol i n h i b i t i o n may be competitive w i t h s u b s t r a t e . (b) I n h i b i t i o n of l l g - H y d r o x y l a t i o n by Metopirone Since the i n t r o d u c t i o n of Metopirone as an i n h i b i t o r of s t e r o i d biogenesis (42), various i n v e s t i g a t i o n s have been conducted on the nature of t h i s i n h i b i t i o n w i t h regard to both s p e c i f i c i t y and mechanism of a c t i o n . I n h i b i t i o n of 116-hy d r o x y l a t i o n by Metopirone has been demonstrated both in -vivo (44) and in vitro (45-49). Studies i n our l a b o r a t o r y on the e f f e c t s of Metopirone on 116-hydroxylation and on the concomitant o x i d a t i o n of NADPH were undertaken, and these e f f e c t s were compared wi t h those of dicumarol. The e f f e c t of Metopirone on NADPH o x i d a t i o n i n the ab-sence of s t e r o i d s ubstrate i s shown i n Figure 40. The rat e of o x i d a t i o n of the coenzyme i s enhanced by Metopirone, the increase being p r o p o r t i o n a l to the q u a n t i t y of Metopirone added. A t h r e e - f o l d increase i n r a t e i s observed i n the - 150 -10 2 0 INCUBATION T IME 3 0 min 4 0 F i g . 40. NADPH o x i d a t i o n a s a f u n c t i o n o f M e t o p i r o n e c o n -c e n t r a t i o n . E a c h r e a c t i o n f l a s k c o n t a i n e d : 0.05 m l o f p r o p y l e n e g l y c o l ; 1.0 m l o f T r i s - M g C l 2 b u f f e r ; 2.0 m l o f enzyme p r e p a r -a t i o n ; a n d 0, 0.18, 0.36, o r 0.72 y m o l e o f M e t o p i r o n e d i s s o l v e d i n 0.1 m l o f T r i s - M g C l 2 b u f f e r . NADPH (0.8 ymole) d i s s o l v e d i n 0.05 m l o f T r i s M g C l 2 b u f f e r was a d d e d a t z e r o t i m e . F i n a l c o n c e n t r a t i o n ( y m o l e s / 1 ) o f M e t o p i r o n e : 0 (©), 55.5 ( o ) , 111 ( A ) , 222 ( A ) . - 151 --4 presence of 2.2 x 10 M Metopirone. The s t i m u l a t i o n of NADPH o x i d a t i o n by Metopirone i s s i m i l a r to that produced by d i -cumarol ( F i g . 36a) i n the absence of deoxycorticosterone, a l -though Metopirone e x h i b i t s a greater s t i m u l a t o r y e f f e c t . D i -- 4 cumarol (1.56 x 10 M) produces a 1.6-fold increase i n NADPH -4 o x i d a t i o n , w h i l e Metopirone (1.1 x 10 M) produces a two-fold i n c r e a s e . P r e l i m i n a r y examination of the i n h i b i t i o n of 116-hydroxy-l a t i o n by Metopirone i n d i c a t e s that t h i s compound e f f e c t i v e l y i n h i b i t s 116-hydroxylation at concentrations much lower than those r e q u i r e d to show a s t i m u l a t i o n of NADPH o x i d a t i o n ( F i g . 41). In the presence of deoxycorticosterone (200 umoles/1), 50% i n h i b i t i o n i s a t t a i n e d at a Metopirone c o n c e n t r a t i o n of 4.2 ymoles/1, a value 2 6 times lower than that r e q u i r e d t o produce a two-fold s t i m u l a t i o n of NADPH o x i d a t i o n . The nature of the i n h i b i t i o n curve obtained w i t h Metopirone d i f f e r s from that obtained w i t h dicumarol ( F i g . 37). Metopirone e f f e c t i v e l y i n h i b i t s 116-hydroxylation at lower concentrations and over a narrower c o n c e n t r a t i o n range of the i n h i b i t o r when compared to i n h i b i t i o n of l l g - h y d r o x y l a t i o n by dicumarol. The e f f e c t of Metopirone on NADPH o x i d a t i o n i n the absence and presence of substrate deoxycorticosterone i s shown i n Figure 42a. Low concentrations of Metopirone produce a s l i g h t increase i n the r a t e of NADPH o x i d a t i o n over t h a t of the c o n t r o l . However, upon a d d i t i o n of deoxycorticosterone, the increase i n NADPH o x i d a t i o n i n the presence of Metopirone i s not as great as i n i t s absence, suggesting an i n h i b i t o r y - 152 -100 2 [METOPIRONE] , M-moles/l F i g . 4 1 . I n h i b i t i o n o f t h e 1 1 6 - h y d r o x y l a t i o n o f d e o x y c o r t i -c o s t e r o n e a s a f u n c t i o n o f M e t o p i r o n e c o n c e n t r a t i o n . I n c u b a t i o n s 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 i n F i g . 4 0 . D e o x y c o r t i c o s t e r o n e - 1 , 2 - 3 H , 0 . 6 7 y m o l e , ( s p e c i f i c a c t i v i t y 2 . 1 9 x 1 0 5 c p m / y m o l e ) was a d d e d d i s s o l v e d i n 0 . 0 5 m l o f p r o p y -lene g l y c o l . A f t e r 5 m i n u t e s p r e - i n c u b a t i o n , NADPH ( 1 . 1 y m o l e s ) was a d d e d d i s s o l v e d i n 0 . 0 5 m l o f T r i s - M g C l 2 b u f f e r . A f t e r 20 m i n u t e s o f i n c u b a t i o n , r e a c t i o n s w e r e t e r m i n a t e d b y t h e a d d i t i o n o f c h l o r o f o r m (5 ml) t o e a c h f l a s k . - 153 -e f f e c t of Metopirone on the h y d r o x y l a t i o n r e a c t i o n . This i n -h i b i t i o n i s confirmed by the data i n Figure 42b showing the r a t e of formation of c o r t i c o s t e r o n e from deoxycorticosterone. In the presence of Metopirone (2.7 ymoles/1, f i n a l concentra-t i o n ) there i s a 46% decrease i n the i n i t i a l r a t e of l l g -h y d r o x y l a t i o n . The s l i g h t s t i m u l a t i o n of NADPH o x i d a t i o n at Metopirone concentrations t h a t e f f e c t i v e l y i n h i b i t l l g — hydroxy-l a t i o n ( F i g s . 42a and 42b) i n d i c a t e s that t h i s i n h i b i t i o n i s not produced by a r a p i d u t i l i z a t i o n of NADPH present i n the r e a c t i o n mixture and t h a t some other mechanism of i n h i b i t i o n must be i n v o l v e d . An examination of the k i n e t i c s of Metopirone i n h i b i t i o n was c a r r i e d out and the r e s u l t s are shown i n Figure 43. In the Dixon p l o t of 1/v vs i, the l i n e s j o i n i n g the experimental values obtained at each substrate c o n c e n t r a t i o n i n t e r s e c t at a p o i n t above the ab c i s s a corresponding t o 1/V m a x determined i n the absence of i n h i b i t o r . The i n t e r s e c t i o n of the l i n e s at t h i s p o i n t e s t a b l i s h e s t h a t Metopirone i n h i b i t i o n i s com-p e t i t i v e i n nature. The p o i n t of i n t e r s e c t i o n a l s o gives the value K^ . of 1.0 x 10 moles/1. The competitive nature of Metopirone i n h i b i t i o n has a l s o been demonstrated by Dominguez and Samuels (45) employing a whole homogenate of r a t adrenal t i s s u e . Thus Metopirone i s a very potent i n h i b i t o r of l l g -h y d r o x y l a t i o n , e f f e c t i v e l y i n h i b i t i n g the r e a c t i o n a t concen-t r a t i o n s much lower than the K m of the subs t r a t e d e o x y c o r t i -costerone (5.5 x 10 ^ moles/1). The high a f f i n i t y of Meto-pirone f o r the l l g - h y d r o x y l a s e may e x p l a i n the f i n d i n g of - 154 -d E o to UJ o < CD CC O 01 CO < i 0 . 8 0 . 4 • 1 0 F i g . 42a INCUBATION TIME , min F i g . 42b F i g . 42a. E f f e c t of Metopirone and deoxycorticosterone on NADPH o x i d a t i o n . Incubations were c a r r i e d out as described i n F i g . 40. Deoxycorticosterone, 0.67 ymole, was added d i s s o l v e d i n 0.05 ml of propylene g l y c o l . Metopirone, 0.013 ymole, was added d i s -solved i n 0.1 ml of Tr'is-MgCl 2 b u f f e r . NADPH (0.8 ymole) was added d i s s o l v e d i n 0.05 ml of T r i s - M g C l 2 b u f f e r at zero time. C o n t r o l (o); plus deoxycorticosterone (©); plus Metopirone (•) ; plus Metopirone and deoxycorticosterone (s) . F i g . 42b. E f f e c t of Metopirone on the H B - h y d r o x y l a t i o n of deox y c o r t i c o s t e r o n e . Incubations were c a r r i e d out as described i n F i g . 40. Deoxycorticosterone-4- 1 "*C, 0.47 ymole, ( s p e c i f i c a c t i v i t y , 6.63 x 10 5 cpm/ymole) was added d i s s o l v e d i n 0.05 ml of propylene g l y c o l . Metopirone, 0.0088 ymole, was added d i s -solved i n 0.1 ml of T r i s - M g C l 2 b u f f e r . A f t e r 5 minutes pre-i n c u b a t i o n , NADPH (1.7 ymoles) was added d i s s o l v e d i n 0.05 ml of T r i s - M g C l 2 b u f f e r . A sample (1.0 ml) was removed from each r e a c t i o n f l a s k a f t e r 4, 8, and 12 minutes of i n c u b a t i o n and p i p e t t e d i n t o chloroform (2 ml) to terminate the r e a c t i o n . C o n t r o l (o), plus Metopirone (©). - 155 -6 . 0 T - 0 . 2 • 0 0.2 0.4 [METOPIRONE] ,/imoles/l F i g . 43. 1/v versus i p l o t of Metopirone i n h i b i t i o n of the 118-hydroxylation of de o x y c o r t i c o s t e r o n e . Each r e a c t i o n f l a s k contained: d e o x y c o r t i c o s t e r o n e - 1 , 2-3H, ( s p e c i f i c a c t i v i t y 7.2 x 10 7 cpm/pmole) d i s s o l v e d i n 0.05 ml of propylene g l y c o l ; 0.70 ml of T r i s - M g C l 2 b u f f e r ; 1.30 ml of enzyme p r e p a r a t i o n ; and Metopirone d i s s o l v e d i n 0.05 ml of T r i s - M g C l 2 b u f f e r . A f t e r 8 minutes p r e - i n c u b a t i o n , NADPH (1.7 umoles) d i s s o l v e d i n 0.05 ml of T r i s - M g C l 2 b u f f e r was added. A sample (1.0 ml) was removed from each r e a c t i o n f l a s k a f t e r 90 seconds of i n c u b a t i o n and p i p e t t e d i n t o e t h y l acetate (2 ml) to terminate the r e a c t i o n . F i n a l c o n c e n t r a t i o n (umoles/1) of deoxy c o r t i c o s t e r o n e -1,2-3H 3.35 (•), 10.2 (©), 30.5 (o). - 156 -Sharma et al (41) th a t Metopirone i n h i b i t i o n of s t e r o i d 118-h y d r o x y l a t i o n could not be r e l i e v e d by i n c r e a s i n g the sub-s t r a t e c o n c e n t r a t i o n . Under t h e i r experimental c o n d i t i o n s , the g r e a t e s t molar r a t i o of deoxycorticosterone:Metopirone examined was 4:1, a r a t i o too low to permit demionstrable r e v e r s a l of i n h i b i t i o n . Of p a r t i c u l a r i n t e r e s t i s the k i n e t i c s obtained at the lowest deoxycorticosterone c o n c e n t r a t i o n , 3.35 umoles/1 ( F i g . 43). At t h i s substrate c o n c e n t r a t i o n , there i s a change i n slope of the curve as the Metopirone concentra-t i o n i s increased. This slope change i s not observed at higher s u b s t r a t e concentrations. This change i n slope i s very s i m i l a r to t h a t obtained w i t h dicumarol ( F i g . 39). However the extent of the slope change i n the presence of Metopirone i s not as prominent as t h a t obtained w i t h dicumarol. The slope change i n the presence of Metopirone i s completely e l i m i n a t e d at a deoxycorticosterone c o n c e n t r a t i o n of 10 umoles/1, w h i l e i n the presence of dicumarol a small change i s s t i l l observed at a deoxycorticosterone c o n c e n t r a t i o n of 35 umoles/1. Furthermore, at low i n h i b i t o r concentrations Metopirone i s a competitive i n h i b i t o r of 118-hydroxylation, w h i l e dicumarol i s a noncompetitive i n h i b i t o r . Thus i t i s u n l i k e l y t h a t these i n h i b i t o r s are a c t i n g by a s i m i l a r mechanism. In both cases however more than one i n h i b i t o r y e f f e c t can be noted, and t h i s second e f f e c t appears to be very s e n s i t i v e to the sub s t r a t e c o n c e n t r a t i o n . - 157 -DISCUSSION I t i s evident from these s t u d i e s t h a t the modes of a c t i o n of dicumarol and Metopirone on 113-hydroxylation are d i f f e r e n t . Dicumarol appears to have two d i s t i n c t i n h i b i t o r y a c t i o n s on 113-hydroxylation. Dicumarol at low con c e n t r a t i o n s , i s a noncompetitive i n h i b i t o r . The i n h i b i t i o n produced at higher dicumarol concentrations i s extremely s e n s i t i v e to the sub-s t r a t e c o n c e n t r a t i o n . Metopirone, on the other hand, i s a competitive i n h i b i t o r of 113-hydroxylation. There are i n d i -c a t i o n s t h a t Metopirone may have more than one i n h i b i t o r y a c t i o n , but t h i s i s apparent only at very low substrate c o n c e n t r a t i o n s . While the k i n e t i c s of i n h i b i t i o n by dicumarol and Meto-pirone are d i f f e r e n t , t h e i r e f f e c t s on NADPH o x i d a t i o n are s i m i l a r . Both dicumarol and Metopirone produce an increase i n the r a t e of NADPH o x i d a t i o n i n the absence of s t e r o i d s u b s t r a t e . This e f f e c t i s more pronounced w i t h Metopirone, a 3.2-fold increase i n NADPH o x i d a t i o n being produced a t a Metopirone conce n t r a t i o n of 222 ymoles/1. Dicumarol, at a con c e n t r a t i o n of 157 ymoles/1, produces a 1.5-fold increase i n NADPH o x i d a t i o n . Both i n h i b i t o r s decrease the NADPH o x i -d a t i o n a s s o c i a t e d w i t h s t e r o i d substrate h y d r o x y l a t i o n . Whether the s t i m u l a t o r y e f f e c t of these compounds on NADPH o x i d a t i o n i s as s o c i a t e d w i t h t h e i r i n h i b i t o r y a c t i o n on 113-hydroxylation i s not c l e a r . I t i s u n l i k e l y t h a t 113-hy d r o x y l a t i o n i n h i b i t i o n r e s u l t s from r a p i d and extensive - 158 -o x i d a t i o n of NADPH by dicumarol or Metopirone. While Meto-pirone can produce a s u b s t a n t i a l increase i n the r a t e of NADPH o x i d a t i o n , t h i s only occurs a t concentrations much higher than t h a t r e q u i r e d to i n h i b i t 113-hydroxylation. Both dicumarol and Metopirone have oxo groups capable of undergoing r e d u c t i o n ( F i g . 3). I t i s p o s s i b l e t h a t the increased r a t e of NADPH o x i d a t i o n observed i n the presence of these compounds i s due to the a c t i o n of a dehydrogenase present i n the enzyme pr e p a r a t i o n . Birmingham and K r a u l i s have observed t h a t the r a t adrenal gland reduces the oxo group of Metopirone to a hydroxyl f u n c t i o n and tha t the reduced d e r i v a t i v e s does not i n h i b i t 113-hydroxylation (161). Metabolites of dicumarol have a l s o been i s o l a t e d from the ur i n e of dicumarol-treated r a t s (162). A l t e r n a t i v e l y these i n h i b i t o r s could be a c t i n g as e l e c t r o n acceptors to an enzyme i n the 113-hydroxylase pathway thus i n t e r r u p t i n g the flow of e l e c t r o n s to the s i t e of deoxycorticosterone h y d r o x y l a t i o n . K i n e t i c examination of dicumarol i n h i b i t i o n of 113-hy d r o x y l a t i o n r e v e a l s t h a t t h i s compound has more than one mode of a c t i o n . At low dicumarol c o n c e n t r a t i o n s , i n h i b i t i o n of 113-hydroxylation i s r e l a t i v e l y i n s e n s i t i v e to v a r i a t i o n s i n dicumarol c o n c e n t r a t i o n and i s noncompetitive w i t h sub-s t r a t e deoxycorticosterone. At high dicumarol c o n c e n t r a t i o n s , i n h i b i t i o n of 113-hydroxylation i s extremely s e n s i t i v e to v a r i a t i o n s i n dicumarol c o n c e n t r a t i o n . In a d d i t i o n , t h i s secondary e f f e c t of dicumarol can be g r e a t l y diminished by i n c r e a s i n g the substrate deoxycorticosterone c o n c e n t r a t i o n - 159 -suggesting a p o s s i b l e competition of substrate and i n h i b i t o r f o r the l l g - h y d r o x y l a s e at the higher dicumarol concentrations. Although n o n l i n e a r k i n e t i c s are o f t e n a s s o c i a t e d w i t h a l l o s t e r i c enzymes (163-167), d e v i a t i o n s from Michaelis-Menton k i n e t i c s need not n e c e s s a r i l y imply: a l l o s t e r i c processes. Gawron et al. (168) suggest t h a t nonlinear k i n e t i c s can be explained i n terms of a mul t i s t a g e r e a c t i o n sequence. Datta and Prakash (169) i n studie s w i t h the aspartokinase of Rhodo-speudomonas sphevoides observed that r e c i p r o c a l p l o t s of 1/v vs 1/[ATP] y i e l d e d b i p h a s i c curves that they a t t r i b u t e d to two b i n d i n g s i t e s on the enzyme molecule f o r ATP. The b i -phasic curves obtained i n the 1/v vs i p l o t ( F i g . 39) demon-s t r a t i n g dicumarol i n h i b i t i o n of l l g - h y d r o x y l a t i o n may l i k e -wise i n d i c a t e two d i s t i n c t i n h i b i t o r y s i t e s on a member enzyme of the l l g - h y d r o x y l a s e pathway f o r t h i s compound. Several explanations of the complex i n h i b i t i o n of l l g -h y d r o x y l a t i o n by dicumarol may be proposed. l l g - h y d r o x y -l a t i o n r e q u i r e s the i n t e r a c t i o n of s t e r o i d s u b s t r a t e , molecular oxygen, and the reducing equivalents of NADPH f o r the i n t r o -d u c t i o n of a hydroxyl f u n c t i o n i n t o the s t e r o i d nucleus. At low c o n c e n t r a t i o n s , dicumarol may a l t e r the i n t e r a c t i o n of these f a c t o r s , perhaps by inducing a conformational change i n the enzyme c a t a l y z i n g t h i s i n t e r a c t i o n . Such an a l t e r a t i o n could lead to a reduced r a t e of l l g - h y d r o x y l a t i o n without a f f e c t i n g the bin d i n g of the s t e r o i d substrates to the enzyme molecule. Such a mechanism: of dicumarol i n h i b i t i o n would be noncompetitive w i t h regard to s t e r o i d s u b s t r a t e , and t h i s i s - 160 -i n d e e d f o u n d t o be t h e c a s e a t l o w d i c u m a r o l c o n c e n t r a t i o n s ( F i g . 3 8 ) . A l t e r n a t i v e l y d i c u m a r o l a t l o w c o n c e n t r a t i o n s c o u l d be a f f e c t i n g t h e b i n d i n g o f m o l e c u l a r o x y g e n t o c y t o -c hrome P-450. A g a i n , i n c r e a s i n g t h e s t e r o i d s u b s t r a t e c o n -c e n t r a t i o n w o u l d n o t o v e r c o m e t h i s i n h i b i t i o n . D a t t a (170) ha s p o i n t e d o u t t h a t i n r e a c t i o n s i n v o l v i n g more t h a n one s u b s t r a t e , m o d i f i e r s may i n f l u e n c e t h e b i n d i n g o f one s u b -s t r a t e a n d n o t i n f l u e n c e t h a t o f t h e o t h e r s . E v i d e n c e now s u g g e s t s t h a t 1 1 6 - h y d r o x y l a t i o n i n v o l v e s t h e s e q u e n t i a l i n t e r a c t i o n o f a t l e a s t f o u r c o m p o n e n t s i n t h e f o r m o f a n e l e c t r o n t r a n s p o r t c h a i n . T h e s e c o m p o n e n t s a r e NADPH, a d r e n o d o x i n r e d u c t a s e , a d r e n o d o x i n , a n d c y t o c h r o m e P-450. I n t e r a c t i o n o f d i c u m a r o l w i t h a component o f t h i s c h a i n p r i o r t o t h a t a c t u a l l y i n v o l v e d i n s t e r o i d b i n d i n g w o u l d r e s u l t i n t h e n o n c o m p e t i t i v e t y p e o f i n h i b i t i o n t h a t i s o b s e r v e d . The s e c o n d a r y i n h i b i t o r y e f f e c t o f d i c u m a r o l a t h i g h e r c o n c e n t r a t i o n s i s s e n s i t i v e t o s t e r o i d s u b s t r a t e ( F i g . 3 9 ) . T h i s s u g g e s t s t h a t a t t h e s e c o n c e n t r a t i o n s , d i c u m a r o l i s i n t e r f e r i n g w i t h t h e a c t u a l b i n d i n g o f d e o x y c o r t i c o s t e r o n e t o t h e 1 1 6 - h y d r o x y l a s e c o m p o n e n t , c y t o c h r o m e P-450. W h e t h e r t h i s s e c o n d a r y i n h i b i t i o n i s c o m p e t i t i v e o r n o n c o m p e t i t i v e i n n a t u r e c a n n o t be u n e q u i v o c a l l y e s t a b l i s h e d b e c a u s e t h i s e f f e c t i s s u p e r i m p o s e d on t h e n o n c o m p e t i t i v e i n h i b i t i o n p r o -d u c e d a t t h e l o w e r d i c u m a r o l c o n c e n t r a t i o n s . The o b s e r v e d i n h i b i t i o n by h i g h e r d i c u m a r o l c o n c e n t r a t i o n s i s o f a m i x e d t y p e ( F i g . 3 8 ) . D i c u m a r o l c o u l d be i n t e r f e r i n g w i t h t h e - 161 -bin d i n g of deoxycorticosterone to cytochrome P-4 50 by i t s e l f b i n d i n g to the sub s t r a t e s i t e , or by inducing a conformational a l t e r a t i o n i n the enzyme t h a t precludes binding of the s t e r o i d s u b s t r a t e . Although the two observed e f f e c t s of dicumarol on 116-h y d r o x y l a t i o n may be separate and d i s t i n c t , the p o s s i b i l i t y e x i s t s t h a t they are i n t e r r e l a t e d . A conformational a l t e r a -t i o n produced by the bi n d i n g of the f i r s t dicumarol molecule may be r e q u i r e d before the second dicumarol molecule can bind to the second s i t e . The mechanism of dicumarol i n h i b i t i o n cannot be c l e a r l y e s t a b l i s h e d without a more d e t a i l e d study of the i n t e r a c t i o n of dicumarol w i t h the components of the 116-hydrOxylase pathway. In c o n t r a s t to dicumarol, Metopirone i s a competitive i n h i b i t o r of 116-hydroxylation ( F i g . 43). Metopirone i n h i b i t s 116-hydroxylation at concentrations lower than the K m of sub-s t r a t e deoxycorticosterone, w h i l e at comparable dicumarol c o n c e n t r a t i o n s , the i n h i b i t i o n of 116-hydroxylation i s l e s s extensive ( F i g s . 37 and 41). The s t r u c t u r a l features of Metopirone t h a t permit com-p e t i t i v e i n h i b i t i o n of deoxycorticosterone 116-hydroxylation are unclear. Dominguez and Samuels (45) suggest t h a t there i s a s t r u c t u r a l r e l a t i o n s h i p between the c o r t i c o s t e r o i d s i d e -chain and Metopirone, and th a t the i n h i b i t o r may be i n t e r -f e r i n g w i t h the i n t e r a c t i o n of the a - k e t o l s i d e c h a i n w i t h the enzyme to which i t binds. However, Sharma et al. (41) have reported t h a t androstenedione, a s t e r o i d without an a - k e t o l - 162 -s i d e c h a i n , i s a competitive i n h i b i t o r of l l g - h y d r o x y l a t i o n of deoxycorticosterone. Thus the importance of the s i d e c h a i n i n the b i n d i n g of deoxycorticosterone to the enzyme or i n the s p e c i -f i c i t y of the l l g - h y d r o x y l a s e i s questionable. The e l u c i d a -t i o n of the s t r u c t u r a l features of Metopirone that permit i n h i b i t i o n of l l g - h y d r o x y l a t i o n must await assignment of the r e q u i s i t e s t r u c t u r a l aspects of the s t e r o i d molecule i n v o l v e d i n enzyme binding and s p e c i f i c i t y of the l l g - h y d o x y l a s e . The nature of competitive i n h i b i t i o n i s u s u a l l y explained i n terms of competition of the i n h i b i t o r and s u b s t r a t e • f o r the same s i t e on the enzyme ( i . e . , i s o s t e r i c i n h i b i t i o n ) . However i t i s known (171) tha t although an i n h i b i t i o n may f o l l o w p r e c i s e competitive k i n e t i c s , t h i s does not n e c e s s a r i l y imply t h a t i t competes w i t h the substrate f o r the same bi n d i n g s i t e . The i n h i b i t o r - b i n d i n g s i t e on the enzyme may be near or may p a r t i a l l y overlap the substrate b i n d i n g s i t e . Hence bind i n g of the i n h i b i t o r may a l t e r the conformation of the a c t i v e s i t e and thus a f f e c t the a f f i n i t y of the subs t r a t e f o r the enzyme (172). Metopirone may f u n c t i o n as a competitive i n h i b i t o r of the l l g - h y d r o x y l a s e i n t h i s manner. The data presented i n Figure 43 show that at low substrate concentrations (3.35 umoles/1) the k i n e t i c curve of Metopirone i n h i b i t i o n i s b i p h a s i c . This may i n d i c a t e t h a t Metopirone i s capable of inducing a conformational a l t e r a t i o n i n the enzyme to which i t binds t h a t r e s u l t s i n an increase i n the e f f e c t i v e -ness of Metopirone as an i n h i b i t o r . As t h i s secondary e f f e c t i s not apparent at higher substrate c o n c e n t r a t i o n , i t i s - 163 -concluded t h a t deoxycorticosterone can prevent t h i s conforma-t i o n a l a l t e r a t i o n w i t h the r e s u l t that normal Michaelis-Menton k i n e t i c are observed. A more d e t a i l e d study of Metopirone i n h i b i t i o n at low substrate concentrations i s r e q u i r e d before the s i g n i f i c a n c e of the b i p h a s i c nature of Metopirone i n h i -b i t i o n at low substrate concentrations can be evaluated. - 164 -PART IV EFFECT OF DICUMAROL AND METOPIRONE ON CYTOCHROMES P-450 AND P-420 Evidence suggests t h a t cytochrome P-450 i s the oxygen-acti-v a t i n g component of mixed f u n c t i o n oxidase systems (67,68,72, 76). This hemoprotein may be r e s p o n s i b l e a l s o f o r substrate binding and a c t i v a t i o n (95-98). The p o s s i b i l i t y t h a t d i -cumarol and Metopirone i n h i b i t i o n of l l g - h y d r o x y l a t i o n i s a r e s u l t of an i n t e r a c t i o n of these compounds wit h cytochrome P-450 was t h e r e f o r e deemed worthy of study. The unique ab-s o r p t i o n spectrum produced on binding of carbon monoxide to cytochrome P-450 permits examination of t h i s hemoprotein without p r i o r extensive p u r i f i c a t i o n . The e f f e c t s of d i -cumarol and Metopirone on cytochrome P-450 were i n v e s t i g a t e d under experimental c o n d i t i o n s s i m i l a r to those of the k i n e t i c , experiments.. RESULTS (a) E f f e c t of Dicumarol on Cytochromes P-450 and P-420 The enzyme pr e p a r a t i o n was incubated i n the presence of dicumarol at two i n h i b i t o r c o n c e n t r a t i o n s , but i n the absence of deoxycorticosterone s u b s t r a t e . One dicumarol c o n c e n t r a t i o n (23 ymoles/1) was i n the r e g i o n of noncompetitive i n h i b i t i o n of l l g - h y d r o x y l a t i o n (Fi g s . 38 and 39) w h i l e the second (116 ymoles/1) was i n the reg i o n producing i n h i b i t i o n of a mixed nature. Incubation c o n d i t i o n s were the same as those employed i n the k i n e t i c s t u d i e s . The r e a c t i o n mixture was pre-incubated - 165 -at 37° C f o r 8 minutes i n the presence of dicumarol. NADPH was then added, and the r e a c t i o n continued f o r an a d d i t i o n a l 90 seconds. The r e a c t i o n mixture was then f r a c t i o n a t e d (at 4° C) w i t h ammonium s u l f a t e and the carbon monoxide d i f f e r e n c e spectrum of the f r a c t i o n p r e c i p i t a t i n g between 20% and 40% s a t u r a t i o n w i t h ammonium s u l f a t e was determined. These spectra are i l l u s t r a t e d i n Figure 44. The spectrum of the c o n t r o l i n c u b a t i o n (no dicumarol) shows the presence of both c y t o -chromes P-450 and P-420. In the presence of the lower dicumarol c o n c e n t r a t i o n (23 umoles/1) the amount of cytochrome P-450 present i n t h i s ammonium s u l f a t e f r a c t i o n i s twice that of the c o n t r o l i n c u b a t i o n . There i s a s l i g h t increase i n cyto-chrome P-420 as w e l l . However, a d d i t i o n of dicumarol at the higher c o n c e n t r a t i o n (116 umoles/1) y i e l d s almost e n t i r e l y cytochrome P-4 20. These r e s u l t s suggest t h a t dicumarol may have more than one e f f e c t on cytochrome P-450. A more exten-s i v e study on the e f f e c t of dicumarol c o n c e n t r a t i o n on c y t o -chromes P-450 and P-420 was c a r r i e d out under the same condi-t i o n s as o u t l i n e d i n Figure 44. The e f f e c t of very low d i -cumarol concentrations on cytochromes P-450 and P-420 i s shown i n Figure 45. At dicumarol concentrations as low as 2.3 umoles/1 there i s a s i g n i f i c a n t increase i n the cyto-chrome P-450 l e v e l s . In a d d i t i o n , a t very low dicumarol c o n c e n t r a t i o n s , 0.23 to 2.3 umoles/1, there i s a l s o an increase i n cytochrome P-420. As the dicumarol c o n c e n t r a t i o n i s i n -creased to 11.6 umoles/1, the cytochrome P-420 content dimin-i s h e s . Although the presence of dicumarol at low concentrations - 166 -i r I 1 1 I I :—1 4 0 0 4 2 0 4 4 0 4 6 0 4 8 0 5 0 0 W A V E L E N G T H , m/i . F i g . 44. Recovery of cytochromes P-450 and P-420 from the m i t o c h o n d r i a l a c e t o n e powder enzyme p r e p a r a t i o n a f t e r i n c u b a -t i o n i n the p r e s e n c e of d i c u m a r o l . A p p r o p r i a t e r e a c t i o n f l a s k s c o n t a i n e d : 0.15 ml of propy-l e n e g l y c o l ; 2.1 ml of T r i s - M g C l 2 b u f f e r ; 3.9 ml o f enzyme p r e p a r a t i o n ; d i c u m a r o l d i s s o l v e d i n 0.15 ml of a d i l u t e a l c o -h o l i c KOH s o l u t i o n . A f t e r 8 minutes p r e - i n c u b a t i o n , NADPH (4.9 umoles) d i s s o l v e d i n 0.15 ml of T r i s - M g C l 2 b u f f e r was added. S o l i d ammonium s u l f a t e t o g i v e 20% s a t u r a t i o n was added a f t e r 90 seconds i n c u b a t i o n and cytochromes P-450 and P-420 were i s o l a t e d and measured as d e s c r i b e d i n the methods. C o n t r o l (--- ); p l u s d i c u m a r o l , 23 ( ) o r 116 ( ) umoles/1, f i n a l c o n c e n t r a t i o n . - 167 -0.08 E o o o < CD o CO < 0.06 O CM c5 0.04r E o o ID" I O in 0.02F-8 16 [DICUMAROL] ,ymoles/1 24 F i g . 45. Recovery of cytochromes P-450 and P-420 from the mi t o c h o n d r i a l acetone powder enzyme p r e p a r a t i o n a f t e r incu-b a t i o n i n the presence of low conc e n t r a t i o n s of dicumarol. Incubations were c a r r i e d out as des c r i b e d i n F i g . 44. Cytochrome P-450 (o), cytochrome P-420 (©). - 168 -leads to an increased recovery of cytochrome P-450, l l g -h y d r o x y l a t i o n i s s t i l l i n h i b i t e d ( F i g s . 37, 38 and 39). As the dicumarol c o n c e n t r a t i o n i s increased above 23 ymoles/1 the cytochrome P-450 content of the i s o l a t e d 20-40% ammonium s u l f a t e f r a c t i o n s decreasesand cytochrome P-420 correspondingly increases ( F i g . 46). At a dicumarol c o n c e n t r a t i o n of 145 ymoles/1, a l l cytochrome P-450 o r i -g i n a l l y present i n the enzyme pr e p a r a t i o n has been removed. The increase i n cytochrome P-420 l e v e l s accompanying the decrease i n cytochrome P-450 i n d i c a t e s t h a t the breakdown of cytochrome P-450 produced by dicumarol r e s u l t s i n the formation:;of cytochrome P-420. Thus dicumarol, at these c o n c e n t r a t i o n s , appears to a l t e r the hydrophobic environ-ment of cytochrome P-450, converting i t to cytochrome P-420. In an attempt to determine whether the two e f f e c t s of d i -cumarol on cytochrome P-450 are r e l a t e d to i t s i n h i b i t i o n of 113-hydroxylation, cytochrome P-450 l e v e l s were measured a f t e r i n c u b a t i o n i n the presence of deoxycorticosterone and/or d i -cumarol ( F i g . 47). In the absence of dicumarol, the a d d i t i o n of deoxycorticosterone (7 or 34 ymoles/1) produces an increase i n cytochrome P-450. The increased cytochrome P-450 content i n the presence of deoxycorticosterone has been p r e v i o u s l y d-eimonstrated ( F i g . 17) and at a concent r a t i o n of 34 ymoles/1 the deoxycorticosterone e f f e c t i s maximal. The a d d i t i o n ' o f dicumarol (23 ymoles/1) produces a f u r t h e r increase i n cyto-chrome P-450. The extent of t h i s increase i n cytochrome P-450 i s e s s e n t i a l l y the same i n the presence of deoxycorticosterone - 169 -0 80 160 240 [DICUMAROL] . M . m o l e s /1 F i g . 46. Recovery of cytochromes P-450 and P-420 from the m i t o c h o n d r i a l acetone powder enzyme p r e p a r a t i o n a f t e r i n c u -b a t i o n i n the presence of high c o n c e n t r a t i o n s of dicumarol. Incubations were c a r r i e d out as de s c r i b e d i n F i g . 44. Cytochrome P-450 (o) , cytochrome P-420 (©) . - 170 -DICUMAROL CONCENTRATION, f iMOLES/ l F i g . 47. Recovery of cytochrome P-4 50 from the m i t o c h o n d r i a l acetone powder enzyme p r e p a r a t i o n a f t e r i n c u b a t i o n i n the presence of dicumarol and deoxycorticosterone. Incubations were c a r r i e d out as described i n F i g . 44. Deoxycorticosterone was added d i s s o l v e d i n 0.15 ml of propy-lene g l y c o l . Deoxycorticosterone f i n a l c o n c e n t r a t i o n (umoles/1): 0 (©) , 7.4 ( A ) , 34 (o) . - 171 -(7 o r 34 ymoles/1) o r i n i t s a b s e n c e . T h i s i n d i c a t e s t h a t t h e e f f e c t s o f d i c u m a r o l a n d d e o x y c o r t i c o s t e r o n e a r e i n d e p e n d e n t . F u r t h e r m o r e , i f t h e s e e f f e c t s a r e p r o d u c e d b y t h e s e compounds b i n d i n g t o c y t o c h r o m e P-450 t h e n t h e y m ust b i n d a t d i f f e r e n t s i t e s o n t h e h e m o p r o t e i n . T h i s f i n d i n g i s c o m p a t i b l e w i t h t h e n o n c o m p e t i t i v e n a t u r e o f i n h i b i t i o n o f 1 1 3 - h y d r o x y l a t i o n a f f o r d e d b y d i c u m a r o l a t t h i s c o n c e n t r a t i o n ( F i g s . 38 and 39) The a d d i t i v e e f f e c t s o f d i c u m a r o l a n d d e o x y c o r t i c o s t e r o n e o n c y t o c h r o m e P-450 a l s o r e v e a l t h a t t h e i n c r e a s e p r o d u c e d by e i t h e r compound a l o n e i s n o t t h e m a x i m a l e f f e c t p o s s i b l e on c y t o c h r o m e P-4 50. As t h e d i c u m a r o l c o n c e n t r a t i o n i s i n c r e a s e d a b o v e 60 y m o l e s / 1 , t h e r e i s a d e c r e a s e i n c y t o c h r o m e P-450 a t b o t h d e o x y c o r t i c o s t e r o n e c o n c e n t r a t i o n s (7 and 34 ymoles/1) s t u d i e d ( F i g . 47). The d i c u m a r o l c o n c e n t r a t i o n t h a t i n i -t i a t e s a d e c r e a s e i n c y t o c h r o m e P-450 i s h i g h e r ( a b o v e 60 ymoles/1) i n t h e p r e s e n c e o f d e o x y c o r t i c o s t e r o n e t h a n i n t h e a b s e n c e ( a b o v e 23 ymoles/1) o f t h i s s t e r o i d . The g e n e r a l p r o -f i l e o f t h e l o s s o f c y t o c h r o m e P-450 i s t h e same i n b o t h t h e p r e s e n c e and a b s e n c e o f d e o x y c o r t i c o s t e r o n e , b u t a t any g i v e n d i c u m a r o l c o n c e n t r a t i o n , t h e p r e s e n c e o f d e o x y c o r t i c o s t e r o n e i n c r e a s e s t h e amount o f c y t o c h r o m e P-4 50 t h a t c a n be r e c o v e r e d . Thus i t a p p e a r s t h a t t h e d e c o m p o s i t i o n o f c y t o c h r o m e P-450 p r o d u c e d b y d i c u m a r o l c o m p e t e s w i t h t h e s t a b i l i z a t i o n o f c y t o c h r o m e P-450 a f f o r d e d b y t h e s t e r o i d s u b s t r a t e . The d a t a o n c y t o c h r o m e P-450 ( F i g . 47) w e r e c o m p a r e d t o t h e k i n e t i c d a t a o n d i c u m a r o l i n h i b i t i o n p r e v i o u s l y d e t e r m i n e d - 172 -(F i g . 39) i n order to a s c e r t a i n whether the b i p h a s i c nature of the k i n e t i c curves were r e l a t e d to the e f f e c t s of dicumarol on cytochrome P-450. I f the decomposition of cytochrome P-450 i s re s p o n s i b l e f o r the second dicumarol i n h i b i t o r y a c t i o n on 113-hydro x,y l a t i o n , then t h i s decomposition would s i g n i f i c a n t l y a l t e r the o v e r a l l r e a c t i o n only when cytochrome P-450 l e v e l s had f a l l e n below those l e v e l s observed i n the absence of d i -cumarol but i n the presence of deoxycorticosterone. Examina-t i o n of the data i n Figure 47 re v e a l s that i n the presence of deoxycorticosterone (7 umoles/1), cytochrome P-450 l e v e l s de-crease to l e s s than that of the c o n t r o l at a dicumarol concen-t r a t i o n above 95 umoles/1, w h i l e at a deoxycorticosterone con-c e n t r a t i o n of 34 umoles/1 t h i s value i s 100 umoles/1. Examina-t i o n of the k i n e t i c data i n Figure 39 re v e a l s t h a t at a deoxy-c o r t i c o s t e r o n e c o n c e n t r a t i o n of 7 umoles/1 the second i n h i -b i t o r y a c t i o n of dicumarol i s f i r s t apparent at a dicumarol c o n c e n t r a t i o n of about 105 umoles/1, and at a deoxycorticosterone c o n c e n t r a t i o n of 35 umoles/1 t h i s value i s 135 umoles/1. Thus there i s a c o r r e l a t i o n between dicumarol concentrations b r i n g -ing about the decomposition of cytochrome P-450 to l e v e l s lower than that of incubations c o n t a i n i n g s t e r o i d alone, and the dicumarol concentrations at which the secondary i n h i b i t o r y e f f e c t on 1 1 3-hydroxylation i s demonstrable. Both the decompo-s i t i o n of cytochrome P-450 and the i n h i b i t i o n of 1 1 3-hydroxyla-t i o n produced at high dicumarol concentrations are s e n s i t i v e to changes i n substrate deoxycorticosterone c o n c e n t r a t i o n sug-g e s t i n g that the two events are r e l a t e d . The i n h i b i t i o n of - 173 -l l g - h y d r o x y l a t i o n at high dicumarol concentrations i s a r e s u l t of decomposition of cytochrome P-450. A time study on the decomposition of cytochrome P-450 i n the presence of dicumarol (23 or 233 ymoles/1) was c a r r i e d out to determine whether the i n h i b i t o r was a f f e c t i n g the r a t e of decomposition of t h i s hemoprotein. The r e s u l t s are shown i n Figure 48. E a r l i e r s t u d i e s had shown that the r a t e of break-down of cytochrome P-4 50 was retarded i n the presence of deoxy-c o r t i c o s t e r o n e ( F i g . 20). This r e s u l t i s confirmed by the data presented i n Figure 48. The presence of deoxycortico-sterone reduces the r a t e of P-450 disappearance by 50%. At the lower c o n c e n t r a t i o n (23 ymoles/1) dicumarol decreases the r a t e of P-450 breakdown, by 19%. This decrease i n the r a t e of breakdown of cytochrome P-450 produced by dicumarol i s not as great as t h a t produced i n the presence of sub s t r a t e deoxy-c o r t i c o s t e r o n e . At high c o n c e n t r a t i o n (233 ymoles/1) dicumarol has an opposite e f f e c t to th a t observed at lower concentra-t i o n s . The r a t e of cytochrome P-450 breakdown i s enhanced by 47% over the c o n t r o l r a t e . The r e s u l t s shown i n Figure 48 permit f u r t h e r explana-t i o n of the cytochrome P-450 l e v e l s i l l u s t r a t e d i n Figure 47. The cytochrome P-450 l e v e l s were measured a f t e r approximately 10 minutes of i n c u b a t i o n at 37° C and are a r e s u l t of the e f f e c t s of deoxycorticosterone and dicumarol on the r a t e of cytochrome P-450 decomposition. Dicumarol at low concentra-t i o n s a l t e r s cytochrome P-4 50 i n some manner which increases i t s s t a b i l i t y . This e f f e c t i s analogous to the deoxycortico-- 174 -INCUBATION TIME , min F i g . 48. E f f e c t of deoxycorticosterone and dicumarol on the r a t e of cytochrome P-450 disappearance. • Appropriate r e a c t i o n f l a s k s contained: deoxycorticosterone d i s s o l v e d i n 0.15 ml of propylene g l y c o l ; 2.1 ml of T r i s - M g C l 2 b u f f e r , 3.9 ml of enzyme p r e p a r a t i o n ; and dicumarol d i s s o l v e d i n 0.15 ml of a d i l u t e a l c o h o l i c KOH s o l u t i o n . A f t e r 0, 4, 8, or 12 minutes of i n c u b a t i o n s o l i d ammonium s u l f a t e to gi v e 20% s a t u r a t i o n was added and cytochrome P-450 then was i s o l a t e d and measured as des c r i b e d i n the methods. C o n t r o l (D) ; plus, d e o x y c o r t i c o s t e r o n e , f i n a l concentra-t i o n , 39 umoles/1 (o); plus dicumarol f i n a l c o n c e n t r a t i o n 23 (©), or 233 (o) umoles/1. - 175 -sterone s t a b i l i z a t i o n of cytochrome P-450 but i s not as pro-nounced. Dicumarol at high concentration, on the other hand, al t e r s cytochrome P-450 i n a manner re s u l t i n g i n an increased i n s t a b i l i t y and rapid degradation to cytochrome P-420. The d i s t i n c t e f f e cts of dicumarol may indicate that this i n h i b i t o r can bind to more than one s i t e on cytochrome P-450, a finding i n agreement with the biphasic i n h i b i t i o n k i n e t i c s previously observed (Fig. 39). The effects of dicumarol on cytochrome P-450 and llg--hydroxylation were also studied employing a l y o p h i l i z e d enzyme preparation from sonicated mitochondria. This preparation has the advantage that cytochrome P^450 measurements.can be made d i r e c t l y on the incubation mixture, eliminating the neces-s i t y for ammonium sulfate f r a c t i o n a t i o n . Moreover - cytochrome P-450 that has been enzymatically reduced with NADPH can be measured d i r e c t l y . The effects of dicumarol on NADPH-reduced cytochrome P-450 may then be compared with those i n the 20-40% ammonium sulf a t e f r a c t i o n with dithionite^-reduced cyto-chrome P-450. The res u l t s are shown i n Figure 49. At a dicumarol concentration of 112 ymoles/1 there i s a s l i g h t decrease i n the absorption maximum at 450 my with a corresponding increase i n absorption at 420 my. When the d i -cumarol concentration i s increased to 224 ymoles/1 the absorp^-tion maximum at 450 my i s decreased by 50%. This i s accompanied by a large increase i n absorption at 420 my. The spectral changes produced by dicumarol indicate that cytochrome p-450 - 176 -u 1 i : i 390 420 450 480 W A V E L E N G T H , m u . F i g . 49. E f f e c t of dicumarol on cytochrome P-450 i n the 116-hydroxylase prepared from s o n i c a t e d adrenal mitochondria. Each cuvette contained: 0.05 ml of propylene g l y c o l ; 1.0 ml of T r i s - M g C l 2 b u f f e r ; 25 mg of l y o p h i l i z e d enzyme p r e p a r a t i o n d i s s o l v e d i n 1.95 ml of a 0.154 M s o l u t i o n of KCl ; and 0, 0.35, or 0.70 ymole of dicumarol d i s s o l v e d i n 0.07 ml of a d i l u t e a l c o h o l i c KOH s o l u t i o n . A f t e r 8 minutes p r e - i n c u b a t i o n at 37° C, NADPH (1.1 ymoles) was added d i s -solved i n 0.05 ml of T r i s - M g C l 2 b u f f e r to both the sample and reference cuvettes and the carbon monoxide d i f f e r e n c e spectrum was determined. C o n t r o l ( ).; plus dicumarol 112 ( ), or 224 ( ) ymoles/1, f i n a l c o n c e n t r a t i o n . - 177 -i n the sonicated enzyme pre p a r a t i o n i s converted to cytochrome P-420 i n the presence of dicumarol, an e f f e c t a l s o observed w i t h the m i t o c h o n d r i a l acetone powder prepa r a t i o n ( F i g . 44). S i m i l a r r e s u l t s to those shown i n Figure 49 are obtained i f cytochrome P-450 i s estimated by chemical r e d u c t i o n with d i t h i o n i t e . The e f f e c t of dicumarol on cytochrome P-450 i n the sonicated mitochondria p r e p a r a t i o n i s l e s s than i t s e f f e c t on the cytochrome P-4 50 i n the acetone powder p r e p a r a t i o n . At a dicumarol c o n c e n t r a t i o n of 224 ymoles/1, the extent of conversion of cytochrome P-450 to cytochrome P-420 i s 50% i n the former p r e p a r a t i o n ( F i g . 49) but 100% i n the l a t t e r ( F i g . 46) . The e f f e c t s of various dicumarol concentrations on c y t o -chromes P-450,> P-420 and l l g - h y d r o x y l a t i o n i n the l y o p h i l i z e d enzyme pr e p a r a t i o n are shown i n Table X I I . Both l l g - h y d r o x y -l a t i o n of deoxycorticosterone and the cytochrome P-450 con-ten t d i m i n i s h as the c o n c e n t r a t i o n of dicumarol i s increased. The decrease i n cytochrome P-4 50 i s accompanied by an increase i n cytochrome P-420. However there i s s t r i c t c o r r e l a t i o n between cytochrome P-4 50 and l l g - h y d r o x y l a t i o n only at the highest dicumarol c o n c e n t r a t i o n t e s t e d (233 ymoles/1) when both l l g - h y d r o x y l a s e a c t i v i t y and cytochrome P-450 have been reduced by 50%. This may i n d i c a t e that at intermediate d i -cumarol con c e n t r a t i o n s , the e f f e c t of the i n h i b i t o r on l l g -h y d r o x y l a t i o n may not be wholly a s c r i b e d to the breakdown of cytochrome P-450. Dicumarol does not produce as extensive an i n h i b i t i o n of l l g - h y d r o x y l a t i o n i n the sonicated m i t o c h o n d r i a l - 178 -TABLE X I I E f f e c t of dicumarol on the 113-hydroxylase a c t i v i t y , cytochrome P-450, and cytochrome P-420 content of the enzyme system pre-pared by s o n i c a t i o n of adrenal mitochondria Dicumarol c o n c e n t r a t i o n (umoles/1) Percent of c o n t r o l 113-hydroxylation Percent of c o n t r o l cytochrome P-450 Percent of c o n t r o l cytochrome P-420 23.3 95.5 100 110 58.1 92.3 88 120 116 68.8 83 190 174 58.3 67 320 233 51.4 50 330 Cytochromes P-450 and P-420 were measured i n incubations c a r r i e d out as described i n F i g . 49. For the measurement of 113-hydroxylase a c t i v i t y each r e a c t i o n f l a s k contained: 0.094 ymole of deoxycorticosterone-1,2- 3H ( s p e c i f i c a c t i v i t y 1.89 x 10 7 cpm/umole) d i s s o l v e d i n 0.05 ml of propylene g l y c o l ; 0.70 ml of Tris-MgCl2 b u f f e r ; 17 mg of l y o p h i l i z e d enzyme powder d i s s o l v e d i n 1.30 ml of a 0.154 M KCl s o l u t i o n ; dicumarol d i s -solved i n 0.05 ml of a d i l u t e a l c o h o l i c KOH s o l u t i o n . A f t e r 8 minutes p r e i n c u b a t i o n , NADPH (1.7 ymoles) d i s s o l v e d i n 0.05 ml of T r i s - M g C l 2 b u f f e r was added. A sample (1.0 ml) was removed from each f l a s k a f t e r 90 seconds of i n c u b a t i o n and p i p e t t e d i n t o e t h y l acetate (2 ml) to terminate the r e a c t i o n . - 179 -pr e p a r a t i o n (Table XII) as i t does i n the acetone powder pre-p a r a t i o n ( F i g . 37). Moreover, no increase i n cytochrome P-450 i s observed at low dicumarol concentrations i n the sonicated mitochondria p r e p a r a t i o n (Table X I I ) . This f i n d i n g , as w e l l as the decreased s e n s i t i v i t y of cytochrome P-450 and l l g - h y d r o x y -l a t i o n to higher dicumarol concentrations i n the sonicated p r e p a r a t i o n when compared to the acetone powder p r e p a r a t i o n , can be best explained by a greater s t a b i l i t y of the hemo-p r o t e i n i n the sonicated p r e p a r a t i o n . I t has been p r e v i o u s l y noted t h a t cytochrome P-450 of the sonicated p r e p a r a t i o n was s t a b l e under i n c u b a t i o n c o n d i t i o n s r e s u l t i n g i n a 64% decrease of t h i s hemoprotein i n the acetone powder pr e p a r a t i o n . Thus any s t a b i l i z i n g e f f e c t of dicumarol at low concentrations would not be evident, and cytochrome P-450 i n the sonicated enzyme pr e p a r a t i o n would be more r e s i s t a n t to decomposition at high dicumarol concentrations. (b) E f f e c t of Metopirone on Cytochromes P-4 50 and P-420 The k i n e t i c s of Metopirone i n h i b i t i o n of l l f t - h y d r o x y l a -t i o n are d i f f e r e n t from those observed w i t h dicumarol. Studies of the e f f e c t of Metopirone on cytochrome P-450 were t h e r e f o r e undertaken to determine whether Metopirone a l s o a f f e c t e d t h i s hemoprotein i n a manner d i f f e r e n t from dicumarol. Previous s t u d i e s revealed that Metopirone i n h i b i t e d 116-hydroxylation at low concentrations ( F i g . 41) and produced a s t i m u l a t i o n of NADPH o x i d a t i o n at high concentrations ( F i g . 40). The e f f e c t of a wide range of Metopirone concentrations - 180 -on c y t o c h r o m e P-450 was t h e r e f o r e e x a m i n e d . The r e s u l t s a r e shown i n F i g u r e 50. A t l o w c o n c e n t r a t i o n s , M e t o p i r o n e p r o -d u c e s a l a r g e i n c r e a s e i n t h e c y t o c h r o m e P-450 r e c o v e r e d a f t e r 8 m i n u t e s o f i n c u b a t i o n . T h i s e f f e c t o f M e t o p i r o n e i s s i m i -l a r t o t h a t o b s e r v e d w i t h d e o x y c o r t i c o s t e r o n e ( F i g . 17) a n d may r e f l e c t t h e c o m p e t i t i v e n a t u r e o f M e t o p i r o n e i n h i b i t i o n o f 1 1 3 - h y d r o x y l a t i o n . As M e t o p i r o n e c o n c e n t r a t i o n s " i n c r e a s e ^ ' t h e r e i s a d e c r e a s e i n t h e amount o f r e c o v e r e d c y t o c h r o m e P-4 50 ( F i g . 50) t o a l e v e l t h a t i s n o t i n f l u e n c e d b y f u r t h e r i n -c r e a s e s i n M e t o p i r o n e c o n c e n t r a t i o n . F i g u r e 50 a l s o shows, t h e c y t o c h r o m e P-420 l e v e l s a t v a r i o u s M e t o p i r o n e c o n c e n t r a -t i o n s a f t e r 8 m i n u t e s o f i n c u b a t i o n a t 37° C. A t l o w c o n c e n -t r a t i o n s o f M e t o p i r o n e , c y t o c h r o m e P-420 d e c r e a s e s , p e r h a p s due t o t h e i n c r e a s e d c y t o c h r o m e P-450 p r e s e n t a t t h e s e i n h i -b i t o r c o n c e n t r a t i o n s . As t h e M e t o p i r o n e c o n c e n t r a t i o n i s i n c r e a s e d , c y t o c h r o m e P-420 l e v e l s r e m a i n l o w a n d c o n s t a n t . A t i m e s t u d y o f t h e r a t e o f d e c o m p o s i t i o n o f c y t o c h r o m e P-450 i n t h e p r e s e n c e o f M e t o p i r o n e was c a r r i e d o u t . M e t o -p i r o n e c o n c e n t r a t i o n s i n t h e r e g i o n o f i n c r e a s e d c y t o c h r o m e P-450 r e c o v e r y ( 3 .5 y m o l e s / 1 ) a n d d e c r e a s e d c y t o c h r o m e P-450 r e c o v e r y (27.2 y m o l e s / 1 ) as i n d i c a t e d i n F i g u r e 50 w e r e s e l e c t e d f o r t h e t i m e s t u d i e s . The 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 u r e 5 1 . The a d d i t i o n o f a s m a l l q u a n t i t y o f M e t o -p i r o n e (3.5 y m o l e s / 1 , f i n a l c o n c e n t r a t i o n ) p r e v e n t s t h e t i m e -d e p e n d e n t l o s s o f c y t o c h r o m e P-450 o b s e r v e d i n t h e a b s e n c e o f t h i s i n h i b i t o r ( F i g . 5 1 a ) . C y t o c h r o m e P-450 l e v e l s a r e h i g h e r i n t h e p r e s e n c e o f M e t o p i r o n e t h a n i n i t s a b s e n c e a t - 181 -8 16 6 0 140 [METOPIRONE] , M.moles/1 2 2 0 F i g . 50. Recovery of cytochromes P-450 and P-420 from the mito-c h o n d r i a l acetone powder enzyme p r e p a r a t i o n a f t e r i n c u b a t i o n i n the presence of Metopirone. Each r e a c t i o n f l a s k contained: 0.15 ml of propylene g l y -c o l ; 2.1 ml of T r i s - M g C l 2 b u f f e r ; 3.9 ml of enzyme p r e p a r a t i o n ; and Metopirone d i s s o l v e d i n 0.15 ml of T r i s - M g C l 2 b u f f e r . S o l i d ammonium s u l f a t e to giv e 20% s a t u r a t i o n was added a f t e r 8 minutes of i n c u b a t i o n and cytochromes P-450 and P-420 were i s o -l a t e d and measured as de s c r i b e d i n the methods. Cytochrome P-450 (o), cytochrome P-420 0 . 0 4 0 . 0 4 - 0 . 0 3 -0 .02 --o.oi-F i g . 51. 4 8 12 0 4 • F i g . 5 i a I N C U B A T I O N T I M E , m i n F i g 5 1 b E f f e c t of Metopirone on the r a t e of cytochrome P-450 disappearance, Incubations were c a r r i e d out as described i n F i g . 50. S o l i d ammonium s u l f a t e was added a f t e r 0, 4, 8, or 12 minutes of i n c u b a t i o n and cytochrome P-450 was i s o l a t e d and measured as described i n the methods. F i g . 51a. C o n t r o l (©);. plus Metopirone, 3.5 ymoles/1, f i n a l c o n c e n t r a t i o n (o). F i g . 51b. Co n t r o l (A); plus Metopirone, 28 ymoles/1, f i n a l c o n c e n t r a t i o n ( A ) . - 183 -a l l i n c u b a t i o n t i m e s . T h e r e f o r e M e t o p i r o n e may be a l t e r i n g c y t o c h r o m e P-450 t o p r e v e n t i t s s p o n t a n e o u s d e c o m p o s i t i o n e i t h e r by i n d u c i n g an a l t e r a t i o n i n t h e h e m o p r o t e i n t o a more s t a b l e c o n f o r m a t i o n , o r by b i n d i n g t o t h e c y t o c h r o m e and t h u s p r o t e c t i n g a l a b i l e g r o u p . The e f f e c t o f M e t o p i r o n e a t t h i s c o n c e n t r a t i o n i s v e r y s i m i l a r t o t h a t o b s e r v e d w i t h d e o x y c o r t i c o s t e r o n e ( F i g . 2 0 ) . A t t h e h i g h e r M e t o p i r o n e c o n -c e n t r a t i o n (27.2 u m o l e s / 1 ) l e s s c y t o c h r o m e P-450 i s r e c o v e r e d a t z e r o t i m e i n t h e p r e s e n c e o f M e t o p i r o n e t h a n i n i t s a b -s e n c e ( F i g . 5 1 b ) . The l o w e r l e v e l o f c y t o c h r o m e P-4 50 r e m a i n s u n a l t e r e d t h r o u g h o u t t h e i n c u b a t i o n p e r i o d . Thus t h e c y t o -chrome P-450 l e v e l s i n t h e c o n t r o l i n c u b a t i o n (no M e t o p i r o n e ) a r e h i g h e r t h a n t h o s e i n t h e p r e s e n c e o f M e t o p i r o n e d u r i n g t h e e a r l y s t a g e s o f i n c u b a t i o n , b u t f a l l b e l o w t h o s e i n t h e p r e s e n c e o f M e t o p i r o n e a t t h e l a t e r s t a g e s o f i n c u b a t i o n , due t o t h e c o n t i n u a l d e g r a d a t i o n o f c y t o c h r o m e P-450 t h r o u g h o u t t h e i n c u b a t i o n p e r i o d . A t h i g h M e t o p i r o n e c o n c e n t r a t i o n s t h e l e v e l o f c y t o c h r o m e P-450 i s n o t a f f e c t e d b y e i t h e r i n c u b a t i o n t i m e ( F i g . 51b) o r by c h a n g e s i n M e t o p i r o n e c o n c e n t r a t i o n i n t h i s r e g i o n ( F i g . 5 0 ) . T h e r e f o r e t h e a d d i t i o n o f l a r g e amounts o f M e t o p i r o n e may p r o d u c e a f u r t h e r a l t e r a t i o n i n c y t o c h r o m e P-450 t o a s e c o n d s t a b l e f o r m . The i n i t i a l l o s s o f h e m o p r o t e i n may i n d i c a t e t h a t d u r i n g t h e c o u r s e o f t h i s a l t e r a t i o n t h e r e i s a v e r y r a p i d l o s s o f a p o r t i o n o f t h e c y t o c h r o m e P-450. A l t e r n a t i v e l y , t h e l o s s o f a b s o r b a n c e a t 450 my may be a t t r i -b u t e d t o a n a l t e r a t i o n o f c y t o c h r o m e P-4 50 t h a t r e s u l t s i n a d e c r e a s e i n i n t e n s i t y o f t h e a b s o r p t i o n maximum a t t h i s w a v e l e n t h . - 184 -B e c a u s e t h e 1 1 3 - h y d r o x y l a s e i n h i b i t i o n by M e t o p i r o n e i s a s s o c i a t e d w i t h l o w c o n c e n t r a t i o n s o f t h i s compound ( F i g s . 41 and 4 3 ) , t h e l o w e r l i m i t o f M e t o p i r o n e p r o t e c t i o n o f c y t o -chrome P-450 was i n v e s t i g a t e d . The r e s u l t s a r e shown i n F i g u r e 52. A m a x i m a l i n c r e a s e i n c y t o c h r o m e P-4 50 r e c o v e r y i s o b s e r v e d a t a M e t o p i r o n e c o n c e n t r a t i o n o f 0.5 u m o l e / 1 a n d a s i g n i f i c a n t i n c r e a s e a t 0.05 u m o l e / 1 . Thus M e t o p i r o n e c a n a l t e r t h i s c y t o c h r o m e a t c o n c e n t r a t i o n s i n t h e r e g i o n o f i t s K^ . ( 0 . 1 umole/1) f o r 1 1 3 - h y d r o x y l a s e i n h i b i t i o n . T h i s s u g g e s t s t h a t t h e e f f e c t o f M e t o p i r o n e on c y t o c h r o m e P-450 i s r e l a t e d t o i t s i n h i b i t o r y a c t i o n on 1 1 3 - h y d r o x y l a t i o n . No p r o t e c t i o n o f c y t o c h r o m e P-420 i s a f f o r d e d b y M e t o p i r o n e a t t h e s e c o n -c e n t r a t i o n s ( F i g . 5 2 ) . The e f f e c t o f M e t o p i r o n e on c y t o c h r o m e P-450 i n t h e p r e s e n c e o f NADPH was n e x t e x a m i n e d . D u p l i c a t e r e a c t i o n m i x -t u r e s c o n t a i n i n g M e t o p i r o n e a t c o n c e n t r a t i o n s o f 0, 1, 5 o r 20 u m o l e s / 1 w e r e p r e - i n c u b a t e d f o r 8 m i n u t e s a t 37° C. To one s e r i e s o f f l a s k s NADPH was a d d e d . A l l f l a s k s w e r e i n -c u b a t e d a t 37° C f o r an a d d i t i o n a l 90 s e c o n d s a n d , i n e a c h i n s t a n c e , t h e c y t o c h r o m e P-4 50 f r a c t i o n was i s o l a t e d . The a d d i t i o n o f NADPH ( F i g . 53) d o e s n o t s i g n i f i c a n t l y i n f l u e n c e t h e e f f e c t o f M e t o p i r o n e on c y t o c h r o m e P-450. S i m i l a r r e -s u l t s h a d b e e n f o u n d p r e v i o u s l y w i t h r e g a r d t o t h e e f f e c t o f NADPH o n d e o x y c o r t i c o s t e r o n e p r o t e c t i o n o f c y t o c h r o m e P-450. The i n t e r - r e l a t i o n s h i p b e t w e e n t h e e f f e c t s o f s u b s t r a t e d e o x y c o r t i c o s t e r o n e a n d M e t o p i r o n e on c y t o c h r o m e P-450 was a s c e r t a i n e d by t h e s i m u l t a n e o u s a d d i t i o n o f t h e two compounds - 18 5 -a. E [METOPIRONE] , M-moles/ I F i g . 52. Recovery of cytochromes P-4 50 and P-420 from the mito-c h o n d r i a l acetone powder enzyme p r e p a r a t i o n a f t e r i n c u b a t i o n i n the presence of low co n c e n t r a t i o n s of Metopirone. Incubations were c a r r i e d out as described i n F i g . 50. Cytochrome P-450 (o) , cytochrome P-420 (©) . - 18G -0.04 [METOPIRONE"] , ymoles/1 F i g . 53. Recovery of cytochrome P-450 from the m i t o c h o n d r i a l acetone powder enzyme p r e p a r a t i o n a f t e r i n c u b a t i o n i n the presence of Metopirone and NADPH. Incubations were c a r r i e d out as described i n F i g . 50. A f t e r p r e - i n c u b a t i o n f o r 8 minutes, NADPH (3 ymoles) d i s s o l v e d i n 0.15 ml of T r i s - M g C l 2 b u f f e r was added to one s e r i e s of r e a c t i o n f l a s k s , 0.15 ml of T r i s - M g C l 2 b u f f e r was added to the other. S o l i d ammonium s u l f a t e was added to each r e a c t i o n f l a s k a f t e r 90 seconds of i n c u b a t i o n and cytochrome P-450 then was i s o l a t e d and measured as described i n the methods. C o n t r o l (o), plus NADPH (©). - 187 -to the reaction mixture. The effects of increasing Metopirone concentrations on cytochrome P-450 i n both the absence and presence of deoxycorticosterone are shown i n Figure 54. Both deoxycorticosterone (Fig. 17) and low concentrations of Meto-pirone (Fig. 52) produce an increase i n cytochrome P-450. These effects are also demonstrated i n Fig . 54 when deoxycor-ticosterone i s added (no Metopirone) or when Metopirone i s added (1 umole/1, no deoxycorticosterone). However simultan-eous addition of deoxycorticosterone (42 umoles/1) and Meto-pirone (1 umole/1) does not produce an increase i n cytochrome P-450 greater than that produced by Metopirone alone. Thus the two effects are not additive and d i f f e r from those ob-served on addition of deoxycorticosterone and low concentra-tions of dicumarol (Fig. 47). Metopirone can eliminate the ef f e c t of deoxycorticosterone on cytochrome P^450 suggesting that the i n h i b i t o r prevents the binding of steroid substrate. This finding concurs with the competitive nature of Metopirone-i n h i b i t i o n of 1 1 3-hydroxylation (Fig. 43). The cytochrome P-450 l e v e l i s increased to a greater extent i n the presence of Metopirone alone (1 umole/1) than i n the presence of deoxy-corticosterone alone (42 umoles/1), a finding that may r e f l e c t the extremely high a f f i n i t y of Metopirone for the 116-hydroxy-_7 lase (K^ . Metopirone = 1.0 x 10 moles/1; deoxycorticosterone — 6 = 5.5 x 10 moles/1). Deoxycorticosterone obviates the de-crease i n cytochrome P-450 that i s observed at higher Meto-pirone concentrations (Fig. 54). If the e f f e c t of high con-centrations of Metopirone on cytochrome P-450 r e f l e c t s the - 188 -E o o If) I o ID LU O < CD OC O CO CD < 0 .04 0 . 02 5 10 15 20 [METOP IRONE ] , ymoles/1 F i g . 54. Recovery of cytochrome P-450 from the mitochondri acetone powder enzyme p r e p a r a t i o n a f t e r i n c u b a t i o n i n the presence of Metopirone and deo x y c o r t i c o s t e r o n e . Incubations were c a r r i e d out as described i n F i g . 50. Deoxycorticosterone (42 ymoles/1, f i n a l concentration) was added d i s s o l v e d i n propylene g l y c o l (0.15 ml). Metopirone (©), Metopirone and deoxycorticosterone (o) - 189 -binding of Metopirone to a second s i t e on the hemoprotein, then i t must be concluded t h a t deoxycorticosterone can pre-vent the bin d i n g of Metopirone to t h i s second s i t e . The e f f e c t of Metopirone on cytochrome P-450 i n the sonicated enzyme pr e p a r a t i o n was examined. The r e s u l t s are shown i n Table X I I I . At no con c e n t r a t i o n does Metopirone a l t e r the amount of NADPH-reducible cytochrome P-450, i n d i -c a t i n g a d i f f e r e n c e i n the s t a b i l i t y of cytochrome P-450 i n the sonicated and acetone powder enzyme pre p a r a t i o n s . As the cytochrome P-450 measured i n t h i s experiment (Table XIII) was reduced e n z y m a t i c a l l y by NADPH, one may conclude that Metopirone does' not a f f e c t the r e d u c t i o n of cytochrome P-450 by NADPH. (c) Substrate and In h i b i t o r - I n d u c e d D i f f e r e n c e Spectra of  Cytochrome P-4 50 Recent stud i e s have revealed that the a d d i t i o n of sub-s t r a t e s to both the microsomal and m i t o c h o n d r i a l hydroxylase systems produce s p e c t r a l changes that can be measured by the technique of d i f f e r e n c e spectrophotometry (86,87,95-98). E v i -dence i n d i c a t e s t h a t t h i s spectrum i s the r e s u l t of i n t e r a c -t i o n and bin d i n g of the subs t r a t e w i t h cytochrome P-450 (98) supporting the p r o p o s i t i o n t h a t cytochrome P-450 i s the s t e r o i d -a c t i v a t i n g as w e l l as the oxygen-activating component of s t e r o i d hydroxylase systems. The a b i l i t y of hydroxylase substrates to induce s p e c t r a l changes was employed to study the i n t e r a c t i o n of deoxycorticosterone, dicumarol, and Metopirone w i t h cytochrome - 190 -TABLE X I I I E f f e c t of Metopirone on cytochromes P-450 and P-420 i n the enzyme system prepared by s o n i c a t i o n of adrenal mitochondria Metopirone c o n c e n t r a t i o n (ymoles/1) A Absorbance 450-500 my (P-450) A Absorbance 420-500 my (P-420) 0 0.030 0.007 0 0.032 0.009 3.4 0.029 • 0.018 6.8 0.033 0.014 6.8 0.031 0. 022 Each cuvette contained: 0.0 2 ml of propylene g l y c o l ; 0.33 ml of T r i s - M g C l 2 b u f f e r ; 9 mg of l y o p h i l i z e d enzyme powder d i s s o l v e d i n 0.65 ml of a 0.154 M s o l u t i o n of KC1; and Metopirone d i s s o l v e d i n 0.02 ml of T r i s - M g C l 2 b u f f e r . A f t e r 8 minutes p r e - i n c u b a t i o n at 37° C, NADPH (0.28 ymole) d i s s o l v e d i n 0.02 ml of T r i s - M g C l 2 b u f f e r was added to both the sample and reference cuvettes. The carbon monoxide d i f f e r e n c e spectrum was then determined. - 191 -P-450. (i) Substrate-induced d i f f e r e n c e spectrum of cytochrome P-450. Because the s p e c t r a l changes induced by s t e r o i d substrates are a r e s u l t of i n t e r a c t i o n between the substrate and c y t o -chrome P-450, the enzyme f r a c t i o n p r e c i p i t a t i n g between 20% and 40% s a t u r a t i o n w i t h ammonium s u l f a t e was employed i n these s t u d i e s . This f r a c t i o n has been shown p r e v i o u s l y to c o n t a i n both cytochrome P-450 and cytochrome P-420 ( F i g . 10b). In a d d i t i o n , the cytochrome P-4 50 i n t h i s f r a c t i o n i s r e l a t i v e l y s t a b l e so that the i n t e r a c t i o n of deoxycorticosterone, Meto-pirone and dicumarol w i t h cytochrome P-4 50 could be measured s p e c t r o p h o t o m e t r i c a l l y without having to consider t h a t any e f f e c t of these compounds was due to s t a b i l i z a t i o n or decom-p o s i t i o n of cytochrome P-450. As a p r e c a u t i o n , spectra were measured at room temperature immediately a f t e r a d d i t i o n of the v a r i o u s agents. The deoxycorticosterone-induced d i f f e r e n c e spectrum- of cytochrome P-450 as a f u n c t i o n of the amount of o r i g i n a l enzyme s o l u t i o n used to prepare the 20-40% ammonium s u l f a t e f r a c t i o n i s shown i n Figure 55. The spectrum has a trough at 420 my and a broad maximum centered at 38 5 my. A s i m i l a r d i f f e r e n c e spectrum w i t h deoxycorticosterone has been observed by Cooper et -al. (86) w i t h an adrenal m i t o c h o n d r i a l prepara-t i o n . In a d d i t i o n , s e v e r a l substrates of the microsomal hydroxylase system induce s i m i l a r s p e c t r a i n adrenal and l i v e r microsomes (8 5,87,9 5-98). 380 4 0 0 4 2 0 4 4 0 4 6 0 4 8 0 5 0 0 WAVELENGTH, F i g . 55. S p e c t r a l changes produced by a d d i t i o n of deoxycorticosterone to the 20-40% ammon-ium s u l f a t e f r a c t i o n of the m i t o c h o n d r i a l acetone powder enzyme p r e p a r a t i o n . The 20-40% ammonium s u l f a t e f r a c t i o n from 2, 4, or 8 ml of enzyme p r e p a r a t i o n was d i s s o l v e d i n 1.6 ml of a s o l u t i o n c o n t a i n i n g Tris-MgCl? b u f f e r (0.6 ml) and 0.154 M KC1 (1.0 ml) and d i v i d e d e q u a l l y between two cuvettes. A f t e r recording the b a s e l i n e (500-375 mu), deoxycorticosterone (84 ymoles/1 f i n a l concentration) was added d i s s o l v e d i n 0.02 ml of propylene g l y c o l to the sample cuvette. The reference cuvette r e c e i v e d 0.02 ml of propylene g l y c o l . The d i f f e r e n c e spectrum was then recorded. 20-40% ammonium s u l f a t e f r a c t i o n from: 2 ( ), 4 ( ), and 8 ( ) ml of enzyme p r e p a r a t i o n . - 193 -The e f f e c t of v a r y i n g deoxycorticosterone concentrations on the induced s p e c t r a l change i s shown i n Figure 56. The i n -t e n s i t i e s of both the 420 my trough and the 385 my peak are p r o p o r t i o n a l to the s t e r o i d c o n c e n t r a t i o n . A p l o t of the change of absorbance (420-403 my or 385-420 my) as a f u n c t i o n of s t e r o i d c o n c e n t r a t i o n gives the normal type of enzyme-subs t r a t e s a t u r a t i o n curve ( F i g . 57). From the Lineweaver-Burk p l o t of these data the s u b s t r a t e c o n c e n t r a t i o n r e q u i r e d f o r half-maximal s p e c t r a l change can be obtained ( F i g . 57). This value has been termed the " s p e c t r a l d i s s o c i a t i o n constant" by Schenkman et at. (97) and i s a measure of the enzyme-sub-s t r a t e complex d i s s o c i a t i o n . The value obtained f o r the deoxycorticosterone-cytochrome P-450 complex i s 7 ymoles/1, a value s i m i l a r to the f o r 116-hydroxylation, suggesting a c l o s e r e l a t i o n s h i p between the s p e c t r a l change observed upon i n t e r a c t i o n of deoxycorticosterone and cytochrome P-450, and the formation of c o r t i c o s t e r o n e from deoxycorticosterone. The same value f o r the s p e c t r a l d i s s o c i a t i o n constant i s ob-t a i n e d using e i t h e r A absorbance (420-403) or A absorbance (385-420), i n d i c a t i n g t h a t the o v e r a l l d i f f e r e n c e spectrum as w e l l as the 420 my trough i s a measure of deoxycorticosterone b i n d i n g to the enzyme. ( i i ) E f f e c t of dicumarol on the substrate-induced d i f f e r -ence spectrum of cytochrome P-450. D i r e c t i n t e r a c t i o n of dicumarol w i t h cytochrome P-4 50 could not be measured because dicumarol i t s e l f absorbed i n - 194 -0 .02 l i i 1 — - — _ — J 1 1 3 8 0 4 0 0 4 2 0 4 4 0 460 4 8 0 WAVELENGTH , F i g . 56. The e f f e c t of deoxyc o r t i c o s t e r o n e c o n c e n t r a t i o n on the magnitude of the s p e c t r a l change produced by a d d i t i o n of deox y c o r t i c o s t e r o n e to the 20-40% ammonium s u l f a t e f r a c t i o n of the m i t o c h o n d r i a l acetone powder enzyme p r e p a r a t i o n . Incubations were c a r r i e d out as des c r i b e d i n F i g . 55. The 20-40% ammonium s u l f a t e f r a c t i o n from 8 ml of enzyme p r e p a r a t i o n was d i s s o l v e d i n 1.6 ml of the T r i s - K C l mixture and d i v i d e d e q u a l l y between two cuv e t t e s . Deoxycorticosterone was added to the sample cuvette d i s s o l v e d i n 0.02 ml of propy-lene g l y c o l . The f i n a l c o n c e n t r a t i o n (umoles/1) of deoxycorticosterone was: 2.44 ( ); 5.49 ( ); 12.2 ( ); or 21.8 ( ). - 195 -2 0 0 -I 6 0 • 0 8 16 24 [ D E O X Y C O R T I C O S T E R O N E ] ( i m o l e s /1 100 [DEOXYCORTICOSTERONE] . ( ^ m o l e s / l ) " F i g . 57. Double r e c i p r o c a l p l o t of the change i n absorbance (385-420 mu) or (420-403 my) as a f u n c t i o n of de o x y c o r t i c o -sterone c o n c e n t r a t i o n . Incubations were c a r r i e d out as described i n F i g . 56. R e c i p r o c a l of absorbance c a l c u l a t e d from the change of absorbance from: 385'to 420 my (o), 403 to 420 (©). - 196 -the r e g i o n of the spectrum (500-375 my) employed to measure these i n t e r a c t i o n s . However the e f f e c t of dicumarol on the s u b s t r a t e d i f f e r e n c e spectrum was examined. Figure 58 shows the e f f e c t of dicumarol at concentrations of 176 and 223 ymoles/1 on the s p e c t r a l changes produced by deoxycorticosterone at concentrations of 7.8 ymoles/1 ( F i g . 58a) and 39 ymoles/1 ( F i g . 58b). At both concentrations d i -cumarol decreases the i n t e n s i t y of both the 420 my trough and the 3 85 my peak, the decrease being p r o p o r t i o n a l to the amount of dicumarol present. The i n f l u e n c e of dicumarol on the d i f f e r e n c e spectrum i s diminished by an increase i n the sub-s t r a t e c o n c e n t r a t i o n . The r e s u l t s suggest t h a t dicumarol e i t h e r i n t e r f e r e s w i t h the b i n d i n g of deoxycorticosterone to cytochrome P-450, or i n t e r f e r e s w i t h the a l t e r a t i o n of the hemoprotein by deoxycorticosterone that produces these s p e c t r a l changes. The cytochrome P-450 content of each of the i n c u -b a t i o n mixtures employed to measure these s p e c t r a l changes was determined. In a l l i n s t a n c e s , the hemoprotein content was the same, i n d i c a t i n g that dicumarol was not a f f e c t i n g the deoxy-c o r t i c o s t e r o n e - i n d u c e d s p e c t r a l changes by causing a degrada-t i o n of cytochrome P-450. The a b i l i t y of deoxycorticosterone to induce s p e c t r a l changes i n cytochrome P-450 i n the presence of i n c r e a s i n g d i -cumarol concentrations i s shown i n Figure 59. Deoxycortico-sterone at concentrations s i m i l a r to those employed i n the i n h i b i t i o n k i n e t i c s t u d i e s ( F i g . 39), was added to r e a c t i o n mixtures c o n t a i n i n g dicumarol at concentrations ranging from 3 8 0 4 0 0 420 4 4 0 4 6 0 3 8 0 4 0 0 420 4 4 0 4 6 0 -WAVELENGTH, F i g . 58a F i g . 58b F i g . 58. E f f e c t of dicumarol on the s p e c t r a l changes produced by a d d i t i o n of d e o x y c o r t i -costerone to the 20-40% ammonium s u l f a t e f r a c t i o n of the m i t o c h o n d r i a l acetone powder enzyme pr e p a r a t i o n . The 20-40% ammonium s u l f a t e f r a c t i o n obtained from 8 ml of enzyme p r e p a r a t i o n was d i s -solved i n a s o l u t i o n made up of 0.56 ml of T r i s - M g C l 2 b u f f e r and 1.0 ml of 0.154 M KC1. D i -cumarol d i s s o l v e d i n 0.04 ml of a d i l u t e a l c o h o l i c KOH s o l u t i o n was added and the mixture was e q u a l l y d i v i d e d between two cu v e t t e s . A b a s e l i n e was recorded. Deoxycorticosterone d i s s o l v e d i n 0.02 ml of propylene g l y c o l was added to the sample cuvette; the reference cuvette r e c e i v e d 0.02 ml of propylene g l y c o l . The d i f f e r e n c e spectrum was then recorded. F i g . 58a. Deoxycorticosterone, f i n a l c o n c e n t r a t i o n , 7.8 ymoles/1. Dicumarol, f i n a l c o n c e n t r a t i o n , ymoles/1: 0 ( ), 176 ( ), 233 ( ). F i g . 58b. Deoxycorticosterone, f i n a l c o n c e n t r a t i o n , 39 ymoles/1. Dicumarol, f i n a l c o n c e n t r a t i o n , ymoles/1: 0 ( ), 176 (--• ), 233 ( ). - 198 -0.03 a E O CM sa-in CD ro UJ o < 03 or o to CD < 0 . 0 2 -[DICUMAROL] , M-moles/l F i g . 59. E f f e c t of dicumarol on the magnitude of the s p e c t r a l change (385-420 my) produced by a d d i t i o n of deoxycorticosterone to the 20-40% ammonium s u l f a t e f r a c t i o n of the m i t o c h o n d r i a l acetone powder enzyme p r e p a r a t i o n . Incubations were c a r r i e d out as described i n F i g . 58. Deoxycorticosterone, f i n a l c o n c e n t r a t i o n , ymoles/1: 7.8 (©) , 39 (o) . - 199 -23 to 233 ymoles/1. I t i s apparent that l i t t l e e f f e c t on the i n t e n s i t y of the s p e c t r a l change produced at e i t h e r d e o x y c o r t i -costerone c o n c e n t r a t i o n (7.8 or 39 ymoles/1) i s observed at dicumarol concentrations below 100 ymoles/1, the range over which dicumarol i s a noncompetitive i n h i b i t o r of 113-hydroxy-l a t i o n (Figs. 38 and 39). At dicumarol concentrations greater than 100 ymoles/1 there i s a decrease i n the magnitude of the s p e c t r a l change induced by deoxycorticosterone at a concen-t r a t i o n of 7.8 ymoles/1; a 50% decrease i s observed at a d i -cumarol c o n c e n t r a t i o n of 233 ymoles/1. This e f f e c t of d i -cumarol i s not so prominent at the higher c o n c e n t r a t i o n of s t e r o i d s u b s t r a t e (39 ymoles/1) i n d i c a t i n g that an increase i n deoxycorticosterone c o n c e n t r a t i o n can overcome the i n h i -b i t o r y e f f e c t of dicumarol. The i n f l u e n c e of dicumarol on these deoxycorticosterone-induced spectra cannot wholly ac-count f o r the degree of i n h i b i t i o n of 113-hydroxylation by dicumarol at high concentrations ( F i g . 39). At a deoxy-c o r t i c o s t e r o n e c o n c e n t r a t i o n of 7.0 ymoles/1, 118^hydroxyla-t i o n i s i n h i b i t e d 79% w h i l e the s p e c t r a l change i s i n h i b i t e d 50%. At a deoxycorticosterone c o n c e n t r a t i o n of 35 ymoles/1, 113-hydroxylation i s i n h i b i t e d 62% w h i l e the s p e c t r a l change i s i n h i b i t e d approximately 20%. Only high concentrations of dicumarol (above 100 ymoles/1) a f f e c t the deoxycorticosterone-induced d i f f e r e n c e spectrum. In a d d i t i o n the e f f e c t of dicumarol can be reduced consider-ably by an increase i n substrate c o n c e n t r a t i o n . Therefore i t i s l i k e l y t h a t the e f f e c t of dicumarol on the substrate-induced - 200 -d i f f e r e n c e spectrum of cytochrome P-450 i s r e l a t e d to the second i n h i b i t o r y a c t i o n of dicumarol on 113-hydroxylation th a t i s ob-served a t high concentrations of t h i s compound ( F i g . 39). ( i i i ) Metopirone-induced d i f f e r e n c e spectrum of cytochrome P-450. U n l i k e dicumarol, Metopirone does not e x h i b i t a bsorption i n the r e g i o n of the spectrum where the deoxycorticosterone e f f e c t s on cytochrome P-450 are measured. Therefore i f there i s a d i r e c t i n t e r a c t i o n between Metopirone and cytochrome P-450, t h i s could be s t u d i e d s p e c t r o p h o t o m e t r i c a l l y . That such an i n t e r a c t i o n does occur i s demonstrated by the Metopirone-induced s p e c t r a l changes shown i n Figure 60. The d i f f e r e n c e spectrum e x h i b i t s an absorption maximum at 422 my and a broad trough centered around 400 my. The c o n c e n t r a t i o n of Metopirone re q u i r e d to induce a s i g n i f i c a n t s p e c t r a l change i s greater than t h a t r e q u i r e d to i n h i b i t 113-hydroxylation. Therefore the Metopirone-ihduced s p e c t r a l change i s l i k e l y r e l a t e d to the secondary e f f e c t on cytochrome P-4 50 produced by high Metopirone concentrations p r e v i o u s l y noted ( F i g . 50), and may a l s o be r e l a t e d to the increased r a t e of NADPH o x i d a t i o n t h a t i s observed at high Metopirone concentrations ( F i g . 40). The s p e c t r a l change induced by Metopirone ( F i g . 60) i s s i m i l a r to those produced by organic solvents i n microsomal preparations described by Imai and Sato (98). They observed t h a t organic solvents such as methanol, ethanol, and 2-propanol, at concen-t r a t i o n s from 3% to 5%, produced d i f f e r e n c e spectra w i t h an - 201 -0.02 I . i 1 1 ' «J 3 8 0 4 0 0 4 2 0 4 4 0 4 6 0 4 8 0 WAVELENGTH , mu, F i g . 60. S p e c t r a l changes produced by a d d i t i o n of Metopirone to the 20-40% ammonium s u l f a t e f r a c t i o n of the m i t o c h o n d r i a l acetone powder enzyme p r e p a r a t i o n . The 20-40% ammonium s u l f a t e f r a c t i o n obtained from 8 ml of the enzyme p r e p a r a t i o n was d i s s o l v e d i n a s o l u t i o n made up of 0.56 ml of T r i s - M g C l 2 b u f f e r and 1.0 ml of 0.154 M K C l . 0.04 ml of propylene g l y c o l was added and the mixture was d i v i d e d e q u a l l y between two cu v e t t e s . A b a s e l i n e (500-375 my) was recorded. Metopirone, d i s s o l v e d i n 0.01 ml of T r i s - M g C l 2 b u f f e r was added to the sample cu v e t t e ; the reference cuvette r e c e i v e d 0.01 ml of T r i s - M g C l 2 b u f f e r . The d i f f e r e n c e spec-trum was then recorded. Metopirone f i n a l c o n c e n t r a t i o n , ymoles/1: '0.1 (-••-), 1.0 ( ), 20 (-.-•-), 200 ( ), 400 ( ). - 202 -absorption maximum at 420 my and a trough at 390 my. No con-v e r s i o n of cytochrome P-450 to cytochrome P-420 took place i n these experiments. The solvent concentrations producing these d i f f e r e n c e spectrum were of the same order of magnitude as the concentrations causing changes i n p r o t e i n conformation through a disturbance of hydrophobic i n t e r a c t i o n s . I t i s q u i t e l i k e l y t h a t the s p e c t r a l changes produced at high Metopirone concen-t r a t i o n s are a r e s u l t of a s i m i l a r e f f e c t . (iv) The e f f e c t of Metopirone on the substrate-induced d i f f e r e n c e spectrum of cytochrome P-450. The e f f e c t of Metopirone on the substrate d e o x y c o r t i c o -sterone-induced d i f f e r e n c e spectrum i s shown i n Figure 61. The presence of Metopirone at a c o n c e n t r a t i o n of 0.1 or 0.2 ymole/1 r e s u l t s i n a diminution of the spectrum i n a manner s i m i l a r to t h a t produced by dicumarol. However, Metopirone i n h i b i t s the substrate d i f f e r e n c e spectrum at much lower con-c e n t r a t i o n s than does dicumarol ( F i g . 58), and at concen-t r a t i o n s lower than those r e q u i r e d f o r Metopirone alone e l i c i t s p e c t r a l changes ( F i g . 60). The concentrations of Metopirone th a t decrease the substrate d i f f e r e n c e spectrum are the same as those t h a t produce an i n h i b i t i o n of l l g - h y d r o x y l a t i o n ( F i g . 43). The e f f e c t of i n c r e a s i n g s u b s t r a t e c o n c e n t r a t i o n i s shown i n Figure 61b and Figure 62. The i n h i b i t i o n of the deoxycor-t i c o s t e r o n e d i f f e r e n c e spectrum by Metopirone can be overcome by an increase i n s t e r o i d s ubstrate c o n c e n t r a t i o n . This i n d i -cates a competition of these two compounds f o r binding to 0 . 02 0 . 0 I u 2 < CO or o </) CO < - 0 . 0 I - 0 0 2 .0 2 3 8 0 4 0 0 4 2 0 4 4 0 4 6 0 3 8 0 4 0 0 4 2 0 4 4 0 4 6 0 F i g . 61a W A V E L E N G T H , mjx, p i g . 61b O CjJ F i g . 61. E f f e c t of Metopirone on the s p e c t r a l changes produced by a d d i t i o n of d e o x y c o r t i -costerone to the 20-40% ammonium s u l f a t e f r a c t i o n of the m i t o c h o n d r i a l acetone powder enzyme p r e p a r a t i o n . The 20-40% ammonium s u l f a t e f r a c t i o n obtained from 8 ml of enzyme p r e p a r a t i o n was d i s -solved i n a s o l u t i o n made up of 0.56 ml of T r i s - M g C l 2 b u f f e r and 1.0 ml of 0.154 M KC1 and the mixture was e q u a l l y d i v i d e d between two cuvettes. Deoxycorticosterone d i s s o l v e d i n 0.02 ml of propylene g l y c o l was added and the d i f f e r e n c e spectrum was determined as des-c r i b e d i n F i g . 55. Metopirone d i s s o l v e d i n 0.01 ml of T r i s - M g C l 2 b u f f e r then was added t o both the sample and reference cuvettes and the d i f f e r e n c e spectrum was again recorded. F i g . 61a. Deoxycorticosterone f i n a l c o n c e n t r a t i o n , 2.4 ymoles/1, Metopirone f i n a l c o n c e n t r a t i o n , ymole/1: 0 (— ), 0.1 ( ), 0.2 ( ). F i g . 61b. Deoxycorticosterone f i n a l c o n c e n t r a t i o n , 21.8 ymoles/1, Metopirone f i n a l c o n c e n t r a t i o n , ymole/1: 0 ( ), 0.1 ( ), 0.2 ( ). - 204 -0 .04 [DEOXYCORTICOSTERONE], M-moles /I F i g . 62. E f f e c t of Metopirone On the magnitude of the spec-t r a l change (385-420 my) produced by a d d i t i o n of d e o x y c o r t i c o -sterone to the 20-40% ammonium s u l f a t e f r a c t i o n of the mito-c h o n d r i a l acetone powder enzyme p r e p a r a t i o n . Incubations were c a r r i e d out as described i n Fig.' 61. Metopirone f i n a l c o n c e n t r a t i o n , ymole/1: 0 (o), 0.1 (©), 0.2 (•) . - 205 -cytochrome P-450, a f i n d i n g i n agreement w i t h the competitive nature of Metopirone i n h i b i t i o n of 116-hydroxylation ( F i g . 43). The potent i n h i b i t o r y a c t i o n of Metopirone i s apparent however, f o r s i g n i f i c a n t i n h i b i t i o n of the deoxycorticosterone-induced d i f f e r e n c e spectrum by Metopirone at a co n c e n t r a t i o n of 0.2 umole/1 s t i l l p e r s i s t s a t the highest deoxycorticosterone con-c e n t r a t i o n ( F i g . 62). These data suggest that i n t e r a c t i o n of e i t h e r Metopirone or deoxycorticosterone w i t h cytochrome P-450 i n t e r f e r e s w i t h the binding of the other to t h i s hemoprotein, and t h a t cytochrome P-450 has a greater a f f i n i t y f o r Meto-pirone than f o r deoxycorticosterone. DISCUSSION The experiments that have been described suggest t h a t the i n h i b i t i o n of 116-hydroxylation by dicumarol and Metopirone i s r e l a t e d to t h e i r e f f e c t s on cytochrome P-450 and on the i n t e r a c t i o n of s t e r o i d s u b s t r a t e w i t h t h i s hemoprotein. Various groups of workers (85-87,95-98) have demonstrated t h a t a d d i t i o n of substrates of both microsomal and m i t o c h o n d r i a l hydroxylases to preparations of these systems causes s p e c t r a l changes i n the so r e t r e g i o n of the spectrum c h a r a c t e r i s t i c of hemoproteins. Imai and Sato have provided evidence suggesting t h a t the s p e c t r a l changes are due to substrate i n t e r a c t i o n w i t h cytochrome P-450 (98), drawing a t t e n t i o n t o the r o l e of t h i s hemoprotein as the s u b s t r a t e - a c t i v a t i n g s i t e i n the hy-d r o x y l a t i o n process. We have demonstrated that d e o x y c o r t i -costerone can produce a s p e c t r a l change i n cytochrome P-450 - 206 -i s o l a t e d from an adrenal m i t o c h o n d r i a l acetone powder. This s p e c t r a l change i s dependent on the amount of both substrate and enzyme present i n the r e a c t i o n mixture. The s i m i l a r i t y of the " s p e c t r a l d i s s o c i a t i o n constant" and the K f o r 118-hy d r o x y l a t i o n of subs t r a t e deoxycorticosterone suggests t h a t the observed s p e c t r a l change i n the presence of subs t r a t e i s r e l a t e d to the h y d r o x y l a t i o n process and that i t i s a measure of the formation of an enzyme-substrate complex. The p r e c i s e mechanism whereby deoxycorticosterone., and other hydroxylase s u b s t r a t e s , induce s p e c t r a l changes i n cyt o -chrome P-450 i s not known. A reasonable hypothesis would be that the subs t r a t e binds to a s p e c i f i c s i t e on the p r o t e i n moiety of cytochrome P-450 causing a conformational a l t e r a t i o n i n the hemoprotein molecule w i t h an accompanying m o d i f i c a t i o n of l i g a n d i n t e r a c t i o n w i t h the heme i r o n of cytochrome P-450. Data obtained by e l e c t r o n paramagnetic resonance spectroscopy suggests t h a t substrate b i n d i n g does modify l i g a n d i n t e r a c t i o n w i t h the heme i r o n of cytochrome P-450 (99,100). The f i n d i n g that d i f f e r e n t s p e c t r a l changes can be produced by d i f f e r e n t substrates (95-98) suggests the presence of more than one bind i n g s i t e on cytochrome P-450, at l e a s t i n the microsomal system. While cytochrome P-420 s t i l l appears to r e t a i n s u b s t r a t e - b i n d i n g c a p a c i t y , the very weak s p e c t r a l change ob-served on s u b s t r a t e - P-420 i n t e r a c t i o n (90,98) suggests a g r e a t l y reduced a f f i n i t y of t h i s cytochrome f o r s u b s t r a t e . Thus the hydrophobic nature of cytochrome P-4 50 i s important - 207 -i n substrate i n t e r a c t i o n w i t h t h i s hemoprotein. The present experiments suggest t h a t the i n t e r a c t i o n of deoxycorticosterone w i t h cytochrome P-450 r e s u l t s i n a s t a b i -l i z a t i o n of the hemoprotein. Thus i f the s p e c t r a l changes produced by i n t e r a c t i o n of deoxycorticosterone w i t h cytochrome P-450 r e f l e c t a conformational change i n t h i s hemoprotein, then the a l t e r e d conformation i s more r e s i s t a n t to decomposition. O f - i n t e r e s t a l s o i s the f i n d i n g t h a t deoxycorticosterone can i n t e r a c t w i t h the o x i d i z e d s t a t e of cytochrome P-450 as evidenced by the s p e c t r a l changes produced i n the absence of any r e -ducing agent. Indeed i t has been reported that t o t a l chemical r e d u c t i o n of the cytochrome e l i m i n a t e s the substrate-induced s p e c t r a l changes (86,87,9 5-98). Cammer and Estabrook have observed however (85) tha t enzymatic r e d u c t i o n of cytochrome P-450 under anaerobic c o n d i t i o n s r e s u l t i n g i n t o t a l r e d u c t i o n of the hemoprotein, does not e l i m i n a t e the deoxycorticosterone-induced s p e c t r a l change i n adrenal mitochondria. They suggest th a t chemical r e d u c t i o n w i t h d i t h i o n i t e modifies d e o x y c o r t i c o -sterone i n t e r a c t i o n w i t h cytochrome P-450 but t h a t t h i s modi-f i c a t i o n does not occur when the hemoprotein i s reduced enzy-m a t i c a l l y v i a the cytochrome P-450 r e s p i r a t o r y c h a i n . In the present s t u d i e s i t i s observed t h a t NADPH does not a l t e r the s t a b i l i z i n g e f f e c t s of deoxycorticosterone or Metopirone on cytochrome P-450. Both dicumarol '.and Metopirone e x h i b i t pronounced e f f e c t s on cytochrome P-450. The evidence t h a t deoxycorticosterone - 208 -interacts with cytochrome P-450 and that this hemoprotein i s both the oxygen-activating and substrate-activating component of 113-hydroxylation leads to the conclusion that the eff e c t s of Metopirone and dicumarol on thi s hemoprotein are related to t h e i r i n h i b i t o r y actions on 118-hydroxylation. The e f f e c t of dicumarol on cytochrome P-450 i s complex; at least two d i s t i n c t e f f e cts are discerned. At low concen-trations dicumarol diminishes the rate of decomposition of cytochrome P-4 50 thus increasing the recovery of this hemo-protein. The p r o f i l e of the increased recovery of cytochrome P-450 i n the presence of low dicumarol concentrations i s very si m i l a r to that observed with deoxycorticosterone. However the protective influence of dicumarol i s not as great as that observed with deoxycorticosterone. Spectrophotometric e v i -dence for the d i r e c t i n t e r a c t i o n of dicumarol with cytochrome P-450 could not be obtained because of the absorption of this compound i n the soret region of the spectrum. The s i m i l a r i t y of the protective influence of dicumarol to that of deoxy-corticosterone implies that the i n h i b i t o r binds to the cyto-chrome and increases i t s s t a b i l i t y . However the protective effects of deoxycorticosterone and dicumarol are additive. Also dicumarol, at concentrations that s t a b i l i z e cytochrome P-450, i s a noncompetitive i n h i b i t o r of llg-hydroxylation. These two facts suggest that dicumarol and deoxycorticosterone are binding to separate s i t e s on the cytochrome and that the binding of one compound does not i n t e r f e r e with the binding - 209 -of the other. This i s supported by the f i n d i n g t h a t low con-c e n t r a t i o n s of dicumarol do not a f f e c t the s p e c t r a l changes produced by the b i n d i n g of deoxycorticosterone to cytochrome P-450. The i n h i b i t i o n of 1 1 3-hydroxylation e x h i b i t e d by d i -cumarol suggests t h a t t h i s compound i n t e r f e r e s w i t h the i n t e r -a c t i o n of the substrates of the hydroxylase system, i . e . , oxygen and deoxycorticosterone, e i t h e r by b i n d i n g to a s i t e on cytochrome P-450 between oxygen and the s t e r o i d or by a l t e r i n g the conformation of the hemoprotein i n such a manner as to reduce the i n t e r a c t i o n of the two substrates and hence r e -duce the r a t e of l l g - h y d r o x y l a t i o n . A l t e r n a t i v e l y the b i n d i n g of dicumarol could be i n h i b i t i n g the attachment of oxygen to the heme p o r t i o n of cytochrome P-450. There i s some evidence th a t at low concentrations dicumarol a l s o p r o t e c t s cytochrome P-420 from decomposition ( F i g . 45). Omura and Sato (89) have demonstrated t h a t the d e s t r u c t i o n of cytochrome P-420 i s accompanied by a l o s s of protoheme i n d i c a t i n g a breakdown of the heme moiety of t h i s cytochrome. Thus dicumarol may be b i n d i n g t o , or i n t e r a c t i n g d i r e c t l y w i t h the heme of cytochrome P-450 i n c r e a s i n g i t s s t a b i l i t y but i n h i b i t i n g i t s b i n d i n g of oxygen. E i t h e r of the above mechanisms f o r dicumarol i n h i -b i t i o n of 1 1 3-hydroxylation would y i e l d noncompetitive k i n e -t i c s w i t h regard to substrate deoxycorticosterone as has been demonstrated. As the dicumarol c o n c e n t r a t i o n i s increased, a second e f f e c t becomes apparent. At high concentrations dicumarol - 210 -increases the r a t e of breakdown, and hence the i n s t a b i l i t y of cytochrome P-450, an e f f e c t d i r e c t l y opposed to th a t observed at low dicumarol c o n c e n t r a t i o n s . Therefore under these c o n d i -t i o n s dicumarol a l t e r s cytochrome P-450 i n such manner th a t the hydrophobic environment around the heme i s no longer main-t a i n e d , w i t h the r e s u l t i n g formation of cytochrome P-420. A s i m i l a r conversion of cytochrome P-450 to cytochrome P-420 can be achieved by a wide v a r i e t y of agents i n c l u d i n g deoxycholate, urea, proteases, phospholipase, s u l f h y d r y l reagents and organic solvents (88-94). Whether the dicumarol-produced conversion of cytochrome P-450 t o cytochrome P-420 proceeds by a primary e f f e c t on hydrophobic i n t e r a c t i o n s of the hemoprotein i n a manner s i m i l a r t o t h a t of detergents or urea, or by a secondary e f f e c t through some conformational change i n the p r o t e i n remains t o be e l u c i d a t e d . The i n h i b i t i o n of 1 1 3-hydroxylation produced at high con-c e n t r a t i o n s of dicumarol i s l i k e l y a r e s u l t of the enhanced conversion of cytochrome P-450 to cytochrome P-420 caused by dicumarol at these c o n c e n t r a t i o n s . Cytochrome P-420 does not f u n c t i o n i n 1 1 3-hydroxylation. The e f f e c t s of high concentra-t i o n s of dicumarol on both 1 1 3-hydroxylation and cytochrome P-450 can be diminished by an increase i n s t e r o i d substrate c o n c e n t r a t i o n . The second i n h i b i t o r y a c t i o n of dicumarol on 1 1 3-hydroxylation can be almost e l i m i n a t e d at high concentra-t i o n s of deoxycorticosterone. Cytochrome P-4 50 r e c o v e r i e s at any given dicumarol c o n c e n t r a t i o n i n the reg i o n where the - 211 -d e s t r u c t i o n of t h i s hemoprotein i s evident are increased i n the presence of deoxycorticosterone, the increase being greater than t h a t observed i n the presence of deoxycorticosterone alone. The dicumarol c o n c e n t r a t i o n at which decomposition of c y t o -chrome P-450 i s i n i t i a t e d i s a l s o higher i n the presence of deoxycorticosterone. Under experimental c o n d i t i o n s where high dicumarol concentrations do not produce conversion of cyto-chrome P-450 to cytochrome P-420, t h i s compound can i n h i b i t the s p e c t r a l change observed on a d d i t i o n of deoxycorticosterone to the cytochrome P-450-containing enzyme f r a c t i o n : . . This i n h i b i t i o n can be diminished by an increase i n s u b s t r a t e con-c e n t r a t i o n . Thus a t high dicumarol concentrations there i s competition between deoxycorticosterone and dicumarol f o r cytochrome P-450 b i n d i n g . This i m p l i e s t h a t these two com-pounds e i t h e r b i n d at the same or overlapping s i t e s , or that the conformational change induced i n the hemoprotein by one compound prevents the b i n d i n g of the other. I f dicumarol does produce such a conformation change i n cytochrome P-450, then i n a d d i t i o n to being unable to bind s u b s t r a t e , the hemopro-t e i n becomes unstable and i s converted to cytochrome P-420. The e f f e c t of Metopirone on cytochrome P-450 i s analo-gous to t h a t produced by deoxycorticosterone and dicumarol at low c o n c e n t r a t i o n s . Low concentrations of Metopirone s t a b i -l i z e the cytochrome r e s u l t i n g i n an increase i n recovery of cytochrome P-4 50 a f t e r i n c u b a t i o n i n the presence of t h i s compound. The c o n c e n t r a t i o n at which Metopirone s t a b i l i z e s - 212 -_7 cytochrome P-450 i s i n the r e g i o n of the K. (1.0 x 10 moles/1) f o r i n h i b i t i o n of l l g - h y d r o x y l a t i o n and i n d i c a t e s t h a t the two processes are r e l a t e d . At t h i s c o n c e n t r a t i o n Metopirone a l s o c o m p e t i t i v e l y i n h i b i t s the deoxycorticosterone-induced spec-t r a l change i n the cytochrome. This e f f e c t i s s i m i l a r to t h a t produced by high dicumarol c o n c e n t r a t i o n s , except t h a t con-v e r s i o n to cytochrome P-420 does not occur. The competitive k i n e t i c s of Metopirone i n h i b i t i o n of the l l g - h y d r o x y l a t i o n of deoxycorticosterone, the s i m i l a r p r o t e c t i v e e f f e c t of these two compounds on cytochrome P-450, and the a b i l i t y of Meto-pirone to i n h i b i t the b i n d i n g of deoxycorticosterone to cytochrome P-450 are a l l c o n s i s t e n t w i t h the hypothesis t h a t e i t h e r the two compounds bind a t the same s i t e on cytochrome P-450, or t h a t the conformational change induced by the b i n d i n g of one compound can c o m p e t i t i v e l y prevent the b i n d i n g of the other. Although no s p e c t r a l i n t e r a c t i o n between c y t o -chrome P-450 and Metopirone at these concentrations can be observed, i t i s p o s s i b l e that the i n t e n s i t y of the s p e c t r a l change i s too low to be detected. An increase i n the Metopirone c o n c e n t r a t i o n produces a f u r t h e r a l t e r a t i o n i n cytochrome P-450. There appears to be a r a p i d conversion of t h i s hemoprotein to a second s t a b l e form. This conversion i s accompanied by an i n i t i a l l o s s of cyto-chrome P-450 as measured by the s p e c t r a l change at 450 my. The r a p i d l o s s of absorbance at t h i s wavelength may i n d i c a t e t h a t higher concentrations of Metopirone a l t e r the conforma-t i o n of cytochrome P-450 i n such a manner as to d i m i n i s h the - 213 -e x t i n c t i o n c o e f f i c i e n t of the carbon monoxide-hemoprotein complex. That Metopirone at these concentrations can produce conformational changes i n cytochrome P-4 50 i s suggested by the a b i l i t y of Metopirone to induce s p e c t r a l changes i n t h i s hemoprotein. The Metopirone-induced d i f f e r e n c e spectrum i s very s i m i l a r to those observed on a d d i t i o n of organic solvents to microsomal cytochrome P-450 (98) at concentrations known to induce changes i n p r o t e i n conformation. This second e f f e c t of Metopirone on cytochrome P-450 occurs at concentrations higher than those r e q u i r e d f o r i n h i b i t i o n of deoxycortico-sterone 113-hydroxylation and may not be r e s p o n s i b l e f o r i t s i n h i b i t o r y a c t i o n . The second e f f e c t may be r e l a t e d to the s t i m u l a t i o n of NADPH o x i d a t i o n observed a t high Metopirone concentrations. The secondary e f f e c t of Metopirone can be prevented by the a d d i t i o n of deoxycorticosterone. Thus what-ever a l t e r a t i o n Metopirone a t high concentrations produces i n cytochrome P-450 cannot take place i n the presence of s t e r o i d s u b s t r a t e . Although the dual e f f e c t s of both dicumarol and Metopirone on cytochrome P-450 have been explained i n terms of two separ-ate e f f e c t s and perhaps two separate b i n d i n g s i t e s on cyt o -chrome P-450 f o r each i n h i b i t o r , the p o s s i b i l i t y e x i s t s that the secondary e f f e c t s of these compounds on cytochrome P-450 are merely extensions of t h e i r i n i t i a l e f f e c t s . Imai and Sato (90) have observed t h a t addition- of excessive amounts of su b s t r a t e a n i l i n e to l i v e r microsomes produces a conver-s i o n of cytochrome P-450 to cytochrome P-42 0. We have not - 214 -observed a s i m i l a r conversion of m i t o c h o n d r i a l cytochrome P-450 i n the presence of deoxycorticosterone. However d i -cumarol and Metopirone may be a c t i n g i n a manner analogous to a n i l i n e . The a d d i t i o n of moderate concentrations of these compounds may modify the cytochrome w i t h a r e s u l t a n t increased-• s t a b i l i t y . On f u r t h e r a d d i t i o n s of these reagents m o d i f i c a -t i o n of the cytochrome becomes i n c r e a s i n g l y more d r a s t i c up to a p o i n t where a new conformation i s assumed. In the case of dicumarol, i n t e g r i t y of the cytochrome P-4 50 s t r u c t u r e can no longer be maintained and t h i s r e s u l t s i n i t s conversion to cytochrome P-420. Thus the f i n d i n g s t h a t both dicumarol and Metopirone appear to have more than one e f f e c t on cytochrome P-450 does not n e c e s s a r i l y imply t h a t there are two d i s t i n c t b i n ding s i t e s on t h i s hemoprotein f o r each i n h i b i t o r . The mechanism of i n h i b i t i o n of dicumarol and Metopirone has been explained on the rsupposition that these compounds can induce a l t e r a t i o n s i n cytochrome P-450, presumably i n i t s con-formation, that r e s u l t i n an i n h i b i t i o n of the h y d r o x y l a t i o n process. Observations by a number of workers support the p o s t u l a t i o n t h a t cytochrome P-4 50 e x i s t s i n more than one con-formation. The s p e c t r a l changes i n the hemoprotein induced by the presence of h y d r o x y l a t i o n substrates or i n h i b i t o r s have been observed i n our experiments and by a number of other groups both i n microsomal (87,95-98) and m i t o c h o n d r i a l (85,86) hydroxylase systems. These changes suggest t h a t substrates produce a conformational change i n cytochrome P-4 50 r e s u l t i n g i n an a l t e r a t i o n of the environment or l i g a n d i n t e r a c t i o n of the heme. Cammer and Estabrook (8 5) have s t u d i e d the i n t e r -- 215 -a c t i o n of su b s t r a t e deoxycorticosterone w i t h m i t o c h o n d r i a l cytochrome P-4 50 i n both the o x i d i z e d and reduced s t a t e s and have obtained s p e c t r a l evidence f o r the t r a n s i t i o n of cytochrome P-450 through s e v e r a l s t a t e s during the course of l l g - h y d r o x y -l a t i o n . Evidence f o r the existence of cytochrome P-450 i n m u l t i p l e forms has been provided by Imai and Sato (102-104) during s t u d i e s on the bin d i n g of e t h y l isocyanide to reduced cytochrome P-450 of r a b b i t l i v e r microsomes. They found t h a t the reduced form of t h i s hemoprotein could bind e t h y l i s o -cyanide to y i e l d a spectrum possessing two absorption maxima in. the sd r e t r e g i o n at 430 my and at 455 my. They found t h a t the heights of these absorption maxima were profoundly i n f l u -enced by pH, i n an interdependent manner. S i m i l a r , but l e s s intense pH-dependent s p e c t r a l changes were observed on bin d i n g of substrate a n i l i n e or other l i p o p h i l i c agents to cytochrome P-450. They concluded t h a t i n the reduced s t a t e , cytochrome P-450 e x i s t s i n two i n t e r c o n v e r t i b l e forms t h a t appear to be i n a pH-dependent e q u i l i b r i u m . While a d d i t i o n of substrates and i n h i b i t o r s ' p r o d u c e s p e c t r a l changes i n cytochrome P-450, the presence of these agents does not, i n the m a j o r i t y of i n s t a n c e s , a l t e r the ab-s o r p t i o n maximum a t 450 my of the carbon monoxide complex. Cammer and Estabrook (85) have observed some deoxycortico-s t e r o n e - a l t e r a t i o n of the carbon monoxide spectrum of cyt o -chrome P-450 i n the v i s i b l e r e g i o n , but reported no change i n the 450 my absorpti o n . Schenkman et at. (9 7) found t h a t c e r -- 216 -t a i n s u b s t r a t e s o f the microsomal h y d r o x y l a s e produce a s h i f t i n the a b s o r p t i o n maximum of the c a r b o n monoxide complex, w h i l e o t h e r s u b s t r a t e s have no e f f e c t . T h e r e f o r e the v a r i o u s forms o f cytochrome P-450 produced by a d d i t i o n of v a r i o u s agents need not d i f f e r markedly i n the a b s o r p t i o n s p e c t r a of t h e i r carbon monoxide complexes . On the b a s i s of e x p e r i m e n t a l data p r e s e n t e d i n t h i s t h e s i s , i t i s proposed t h a t d e o x y c o r t i c o s t e r o n e , M e t o p i r o n e , and d i -cumaro l can induce c o n f o r m a t i o n a l changes i n cytochrome P -450. W h i l e these changes do not a l t e r the spectrum of the c a r b o n monoxide complex o f cytochrome P-450, they do a f f e c t the s t a b i l i t y of t h i s hemoprote in and i t s a b i l i t y to f u n c t i o n i n 1 1 6 - h y d r o x y l a t i o n . A summary of the i n t e r a c t i o n s i s p r e s e n t e d i n F i g u r e 63. Cytochrome P-4 50 i s o l a t e d from the acetone powder o f a d r e n a l m i t o c h o n d r i a i s r e l a t i v e l y u n s t a b l e and undergoes spontaneous d e c o m p o s i t i o n to cytochrome P-420 . S t e r o i d sub-s t r a t e s such as d e o x y c o r t i c o s t e r o n e , and low c o n c e n t r a t i o n s o f b o t h d i c u m a r o l and M e t o p i r o n e b i n d to cytochrome P-4 50 p r o d u c i n g a c o n f o r m a t i o n a l change to P L 450 w i t h i n c r e a s e d s t a b i l i t y . The b i n d i n g o f M e t o p i r o n e and d e o x y c o r t i c o s t e r o n e to cytochrome P-450 i s c o m p e t i t i v e i n n a t u r e and t h i s r e s u l t s i n a c o m p e t i t i v e i n h i b i t i o n of 116 - h y d r o x y l a t i o n by M e t o p i r o n e . The c o m p e t i t i v e b i n d i n g of M e t o p i r o n e and d e o x y c o r t i c o s t e r o n e to cytochrome P-450 sugges ts t h a t the two compounds may e i t h e r b i n d to the same s i t e or t h a t the c o n f o r m a t i o n change induced Adrenal M i t o c h o n d r i a l Acetone powder 0.154 M KCl P-420 phospholipid Deoxycorticosterone Dicumarol Metopirone Dicumarol [P-450] ^ ' ' =r±= [P'-450]-:^ = Deoxycorticosterone Deoxycorticosterone Metopirone P-420 [P'''-450] i H I [P''-450] F i g . 63. Schematic r e p r e s e n t a t i o n of the conversion of cytochrome P-450 to d i f f e r e n t conformational s t a t e s by i n t e r a c t i o n w i t h deoxycorticosterone, Metopirone, and d i -cumarol. - 218 -by the binding of e i t h e r compound prevents the bin d i n g of the second compound. Dicumarol binding at low concentrations to cytochrome P-4 50 i s noncompetitive w i t h deoxycorticosterone as the s t a b i l i z i n g e f f e c t s of these two compounds are a d d i t i v e . Therefore they must bind a t d i f f e r e n t s i t e s . The bin d i n g of dicumarol r e s u l t s i n noncompetitive i n h i b i t i o n of l l g - h y d r o x y -l a t i o n suggesting t h a t the b i n d i n g of t h i s i n h i b i t o r i n t e r -f e r e s w i t h the i n t e r a c t i o n of deoxycorticosterone and oxygen, the substrates f o r l l g - h y d r o x y l a t i o n . While the scheme o u t l i n e d i n Figure 63 i n d i c a t e s t h a t deoxycorticosterone, dicumarol, and Metopirone convert c y t o -chrome P-450 t o a more s t a b l e form, cytochrome P'-450, i t i s not meant t o suggest t h a t the b i n d i n g of each of the compounds produces the same conformational change i n the hemoprotein. I t i s q u i t e l i k e l y t h a t deoxycorticosterone, dicumarol, and Metopirone each modify cytochrome P-4 50 i n a d i f f e r e n t manner. The term cytochrome P'-450 i s used i n Figure 63 merely to denote th a t the b i n d i n g of these compounds produces a more s t a b l e form of cytochrome P-450. High concentrations of Metopirone r e s u l t i n a f u r t h e r a l t e r a t i o n of cytochrome P-450 to a form designated, P''-450. This form i s a l s o s t a b l e but a l o s s of some cytochrome P-450 occurs during conversion to t h i s second form. I t i s a l s o pos-s i b l e that the carbon monoxide complex of the cytochrome P 1'-450 has a diminished e x t i n c t i o n c o e f f i c i e n t when compared to th a t of the carbon monoxide complex of cytochrome P'-450. For-- 219 -mation of P''-450 i s prevented by the presence of d e o x y c o r t i -costerone . High concentrations of dicumarol r e s u l t i n the formation of P 1''-450 which i s extremely unstable and very r a p i d l y con-v e r t e d to cytochrome P-420. The binding of dicumarol at high concentrations appears to be competitive w i t h deoxycortico-sterone; hence the formation of P'''-450 i s diminished by a d d i t i o n of the s t e r o i d s u b s t r a t e . The competitive b i n d i n g of dicumarol and deoxycorticosterone to cytochrome P-450 suggests that the second i n h i b i t o r y e f f e c t of dicumarol on l l g - h y d r o x y -l a t i o n at high i n h i b i t o r concentrations i s competitive i n nature. The p h o s p h o l i p i d f r a c t i o n of the acetone e x t r a c t of adrenal mitochondria a l s o has the a b i l i t y to s t a b i l i z e c yto-chrome P-450, as has been p r e v i o u s l y discussed. I t i s not known whether t h i s i s a general e f f e c t due to an increase i n the hy-drophobic nature of the environment of cytochrome P-4 50 or to a s p e c i f i c b i nding of phospholipid w i t h a r e s u l t i n g confor-mational a l t e r a t i o n . Evidence a l s o i n d i c a t e s t h a t the phospho-l i p i d f r a c t i o n may mediate some form of r e c o n s t i t u t i o n of cy-tochrome P-450 from cytochrome P-420 presumably due to a r e -combination of l i p i d w i t h the hemoprotein moiety. I t i s apparent t h a t the s t r u c t u r e of cytochrome P-450 that gives t h i s hemoprotein i t s unusual p r o p e r t i e s i s main-t a i n e d by hydrophobic bonding w i t h i n the cytochrome complex. Deoxycorticosterone, dicumarol, Metopirone, and phospholipid a l l , a p p e a r to exert some i n f l u e n c e on the hydrophobic nature - 220 -of t h i s hemoprotein. The s t r u c t u r a l features of d e o x y c o r t i -costerone, Metopirone, and dicumarol t h a t a l l o w these com-pounds to bind to cytochrome P-450 are as yet unclear. The substrates f o r both the microsomal and m i t o c h o n d r i a l hydroxy-la s e systems are g e n e r a l l y l i p o p h i l i c i n nature. Imai and Sato (104) have s t a t e d t h a t only l i p o p h i l i c agents appear to cause s p e c t r a l changes i n cytochrome P-450 a s s o c i a t e d w i t h t h e i r b i n d i n g . Deoxycorticosterone, Metopirone and dicumarol a l l are c y c l i c compounds possessing l i p o p h i l i c p r o p e r t i e s . Yet i t i s u n l i k e l y t h a t t h i s general property i s r e s p o n s i b l e f o r t h e i r b i n d i n g to t h i s cytochrome. C o r t i c o s t e r o n e and adrenosterone a l s o are l i p o p h i l i c , yet they have no a b i l i t y t o s t a b i l i z e cytochrome P-450. Therefore the i n t e r a c t i o n s of deoxycorticosterone, Metopirone, and dicumarol w i t h cytochrome P-450 e x h i b i t some degree of s p e c i f i c i t y . The s t r u c t u r a l features of Metopirone and dicumarol that permit these compounds to compete w i t h deoxycorticosterone f o r b i n d i n g to cytochrome P-450 are u n c l e a r . While these com-pounds are a l l c y c l i c , they are not s t r u c t u r a l l y s i m i l a r . I t i s t h e r e f o r e u n l i k e l y t h a t they are binding to the same s i t e on the hemoprotein. I t must be concluded that cytochrome P-450 i s complex and possesses m u l t i p l e s i t e s f o r the b i n d i n g of such compounds. The b i n d i n g of one compound must a l t e r the conformation of the binding s i t e s of the other compounds i n such a manner as to prevent t h e i r b i n d i n g to the hemoprotein. The u l t i m a t e e l u c i d a t i o n of the mechanism of i n h i b i t i o n by dicumarol and Metopirone must await an understanding of - 221 -the complex nature of cytochrome P-450. However a l l attempts to p u r i f y t h i s hemoprotein have been unsuccessful due to i t s i n s t a b i l i t y . We suggest that s t u d i e s on the e f f e c t s of sub-s t r a t e s and i n h i b i t o r s of the 113-hydroxylase on cytochrome P-450 and 113-hydroxylation may give much inf o r m a t i o n as to the c h a r a c t e r i s t i c s of the substrate a c t i v e s i t e and the i n -h i b i t o r s i t e s on cytochrome P-450, and the r e l a t i o n s h i p s between these s i t e s . 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