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Oxidation and reduction of the C-11 position of corticosterone and 11-dehydrocorticosterone by mouse… Lewis, Elizabeth Cairine 1962

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OXIDATION AND REDUCTION OF THE C - l l POSITION OF CORTICOSTERONE AND  11-DEHYDROCORTICOSTERONE BY MOUSE LIVER FRACTIONS IN VITRO  by  ELIZABETH CAIRINE LEWIS  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE  REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  i n t h e Department of BIOCHEMISTRY  We a c c e p t t h i s required  THE  thesis  as conforming  t o the  standard  UNIVERSITY OF BRITISH COLUMBIA April,  1962  In presenting  this thesis i np a r t i a l fulfilment of  the r e q u i r e m e n t s f o r a n advanced degree a t t h e U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t t h e 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 . for extensive  I f u r t h e r agree t h a t p e r m i s s i o n  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 p u r p o s e s may be  g r a n t e d by t h e Head o f my Department o r by h i s  representatives.  I t i s understood t h a t copying 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 n o t be a l l o w e d w i t h o u t my w r i t t e n  Department The U n i v e r s i t y o f B r i t i s h Columbia, Vancouver 8, Canada. Date  ^f/^Jb  permission.  A B S T R A C T  OXIDATION AND AND  REDUCTION OF THE  C - l l POSITION OF CORTICOSTERONE  11-DEHTDROCORTICOSTERONE BY MOUSE LIVER FRACTIONS IN VITRO C o r t i c o s t e r o n e and  20a  dihydrocorticosterone  have been found i n the l i v e r and blood of mice i n j e c t e d i n travenously with C-ll-dehydrocorticosterone.  Within a  few  minutes a f t e r i n j e c t i o n the l a t t e r i s no longer d e t e c t a b l e i n the blood and  liver, ( l ) . The  enzyme r e s p o n s i b l e f o r the r e d u c t i o n of the  C - l l keto group has been i s o l a t e d i n the microsomal f r a c t i o n of mouse l i v e r homogenate.  T h i s f r a c t i o n was p r e c i p i t a t e d  by u l t r a c e n t r i f u g a t i o n a t 100,000 x G of the a f t e r c e n t r i f u g a t i o n a t 6,000 x NADPH2 was  supernatant  G.  e s t a b l i s h e d as the coenzyme r e q u i r e d  although l i m i t e d a c t i v i t y could be demonstrated with The  r e a c t i o n can be reversed by use  NAD^.  of NADP.  C e r t a i n k i n e t i c p r o p e r t i e s of the r e a c t i o n have been i n v e s t i g a t e d .  The  r e a c t i o n v e l o c i t y i s p r o p o r t i o n a l to  enzyme c o n c e n t r a t i o n andl l i n e a r with time over the p e r i o d studied. sterone  For the r e d u c t i o n r e a c t i o n with as substrate. K  umoles/ml/min. costerone  m  x 10  -5  and V  „ = 1.6 max  x  For the dehydrogenation r e a c t i o n with  as substrate K  (i.moles/ml/min.  = 1.8  11-dehydrocortico-  m  = 1.7  x 10  -4  and V  „ = 2.7 max  10  -4  cortix  10  -3  - iii  -  A C K N O W L E D G M E N T S  The author wishes t o express her s i n c e r e thanks t o Dr. Marvin Darrach f o r h i s advice and encouragement.  The a s s i s t a n c e of the N a t i o n a l Research Council  i n the form of a Studentship i s g r a t e f u l l y  acknowledged.  - iv-  TABLE OF CONTENTS Page Title  i  Abstract  i i  Acknowledgments  i i i  Table of Contents  iv  Introduction  1  Experimental  11  Results  13  Isolation  of 11(3 reductase a c t i v i t y  Reversibility  of the 118 reductase system  Substrate s p e c i f i c i t y of f r a c t i o n  P^  13 14 14  Coenzyme dependence of crude l i p reductase of mouse l i v e r K i n e t i c s of the 116 reductase system  14 16  Discussion  26  Summary  31  Bibliography  32  - V -  LIST OF TABLES AND FIGURES Page Table I  -  ll-Dehydrocorticosterone-118-Reductase A c t i v i t y i n Mouse L i v e r  15  Fractions.  Table I I - Reduction and Dehydrogenation a t C - l l of Various C o r t i c o s t e r o i d s  by  17  Fraction  of Mouse L i v e r . Fig. I  - E f f e c t of Enzyme C o n c e n t r a t i o n on R e a c t i o n Velocity  (Substrate  20  11-Dehydrocortico-  sterone). Fig. II  - E f f e c t of Time on the Y i e l d of C o r t i c o -  21  sterone. F i g . I l l - E f f e c t of Substrate C o n c e n t r a t i o n on Reaction Velocity  22  (Substrate 11-Dehydrocorti-  costerone). F i g . IV  - E f f e c t of Enzyme Concentration on R e a c t i o n Velocity  Fig. V  23  (Substrate C o r t i c o s t e r o n e )  - E f f e c t of Time on the Y i e l d of 11-Dehydro-  24  corticosterone. F i g . VI  - E f f e c t of Substrate C o n c e n t r a t i o n on Reaction Velocity  (Substrate C o r t i c o s t e r o n e )  25  I N T R O D U C T I O N The ministered  high hepatic  hormones f i r s t  concentration  of exogenously ad-  suggested the importance of the  l i v e r i n s t e r o i d metabolism, i n c l u d i n g t h e i r a c t i v a t i o n , degradation,  and e x c r e t i o n .  Metabolism by the l i v e r o f the  a d r e n o c o r t i c a l hormones has been demonstrated both i n v i v o and  in vitro.  The r e s u l t s have r e v e a l e d t h a t C-21 s t e r o i d s  are a l t e r e d by o x i d a t i o n or r e d u c t i o n at C - l l , 4  reduction of  the s i d e chain, and r e d u c t i o n of the A -3 Keto system of r i n g A.  The 17-keto d e r i v a t i v e s have been found as c o r t i -  c o s t e r o i d metabolites  but these are not major end products.  (2). The  demonstration t h a t c o r t i s o n e i s converted t o  C o r t i s o l i n vivo f i r s t  suggested r e d u c t i o n  of the C - l l  ketone as r e s p o n s i b l e f o r some of the a c t i v i t y a t t r i b u t e d to c o r t i s o n e .  ( 3 ) . Hechter p o i n t e d  though c o r t i s o n e i s r a p i d l y destroyed a c t i v i t y following administration some time. of  previously out t h a t a l -  by the l i v e r , metabolic  of the hormone p e r s i s t s f o r  He, t h e r e f o r e , p o s t u l a t e d t h a t c e r t a i n  metabolites  c o r t i s o n e formed i n the l i v e r are p h y s i o l o g i c a l l y a c t i v e .  (4).  Ingle observed that c o r t i s o n e was unable t o exert i t s  usual e f f e c t on carbohydrate  tolerance  i n the hepatecto-  mized r a t , although a d m i n i s t r a t i o n of an a d r e n o c o r t i c a l ext r a c t produced the normal e f f e c t of a decrease i n glucose tolerance.  I t seemed i n d i c a t e d , t h e r e f o r e , that  cortisone  was a l t e r e d by passage through the l i v e r and the accompanying  -  2 -  changes i n t h e m o l e c u l e must be n e c e s s a r y b e f o r e affect  carbohydrate metabolism.  vations  substantiated  activity  the idea that  A l lthese  The  some o f t h e m e t a b o l i c  to C o r t i s o l .  original biological  findings with  respect to  e x c e p t i o n a l l y l a r g e d i f f e r e n c e between t h e a c t i v i t i e s  of 2 - m e t h y l c o r t i s o n e and 2 - m e t h y l c o r t i s o l reduction  hypothesis,  cortisone being  support the C - l l  t h e low a c t i v i t y p o s s e s s e d by 2 - m e t h y l -  due t o t h e s m a l l  amount o f 2 - m e t h y l c o r t i s o l  w h i c h i s f o r m e d f r o m i t . ( 6 ) . The C - l l k e t o n i c been d e m o n s t r a t e d t o be l e s s a c t i v e t h a n t h e lip  obser-  p r e v i o u s l y a t t r i b u t e d t o c o r t i s o n e may i n f a c t be  d e p e n d e n t on i t s c o n v e r s i o n  the  (5).  i t can  corresponding  hydroxy form f o r s e v e r a l p a i r s of s t e r o i d s .  claimed  therefore  genated adrenal  that biological  activity  steroids i s confined  form has  It is  o f t h e 11-oxy-  t o t h e 11{3 h y d r o x y  f o r m s e x c e p t when t h e C>,11, k e t o n e s c a n be r e d u c e d t o t h e former. The duction  that tissues vary  in ability  to effect r e -  o f t h e C - l l k e t o p o s i t i o n was d e m o n s t r a t e d when t h e  antirheumatic injection (7).  fact  This  metabolite  effect  i n t o the j o i n t  could  space w h i l e  i s i n keeping with  be o b s e r v e d on d i r e c t  c o r t i s o n e was i n a c t i v e .  the previous  ideas  as t h e a c t i v e f o r m s i n c e i f c o r t i s o n e  into the c i r c u l a t i o n seems t h a t  of C o r t i s o l  there  i ti s fully  i s a discrepancy  active.  on a  liver  i s injected  Therefore, i t  between t h e i n v i v o  f i n d i n g s t h a t c o r t i s o n e and periods  of time and  c o r t i c o s t e r o n e are a c t i v e over  the i n v i v o and  in vitro  observations  t h a t C - l l keto compounds are r a p i d l y metabolized by l i v e r to a v a r i e t y of products.  The  the  best e x p l a n a t i o n  seems  to be t h a t some of the products formed i n l i v e r metabolism possess the a n t i - a r t h r i t i c , t h y m o l y t i c , a c t i v i t i e s a s s o c i a t e d with c o r t i s o n e  and  biological  administration.  Hechter, Frank, Caspi and Frank (8) Schneider and Horstman (9)  other  as w e l l  as  have demonstrated C o r t i s o l to  metabolized by the l i v e r as r a p i d l y as c o r t i s o n e .  be  However,  some b i n d i n g of the a c t i v e form to a t i s s u e c o n s t i t u e n t  could  e x p l a i n the prolonged a c t i v i t y d e s p i t e the r a p i d i t y of  hepatic  a l t e r a t i o n s i n the molecule.  Axelrod  and M i l l e r  t h a t i n order f o r c o r t i s o n e or C o r t i s o l to exert  postulated their  a c t i o n , the a c t i v e form of the hormone must be bound to hepatic  t i s s u e i n order  to prevent d e s t r u c t i o n by the  through subsequent metabolic changes. ( 3 ) .  Binding  non-  liver  of s t e r o i d  to plasma p r o t e i n , p a r t i c u l a r l y to the g l o b u l i n f r a c t i o n i n human plasma has  been shown. ( 1 0 ) .  hormones, C o r t i s o l and  The  active corticosteroid  c o r t i c o s t e r o n e , c i r c u l a t e i n the  plasma l a r g e l y as a complex formed with an a g l o b u l i n c a l l e d the c o r t i c o s t e r o i d b i n d i n g g l o b u l i n . where the b i n d i n g  bind-  other plasma p r o t e i n s i s observed,  (ll).  I t i s now  w e l l e s t a b l i s h e d t h a t the subsequent r e -  of r i n g A and  responsible  concentrations  s i t e s of t h i s p r o t e i n are s a t u r a t e d ,  i n g by albumin and  ductions  At  f o r the  the  side chain of the molecule are  e l i m i n a t i o n of b i o l o g i c a l a c t i v i t y .  (12).  - 4 In f a c t , a l l p o s t u l a t e d metabolic  schemes show r e d u c t i o n to  be the major i n a c t i v a t i n g mechanism f o r adrenal s t e r o i d s i n the l i v e r and  the c h i e f end  cortical  products f o r such  metabolism to be s a t u r a t e d polyhydroxylated  C-21  steroids.  (2,13,14). I t was  p o s t u l a t e d by Hubener, Fukushima and  Gallagher  that the r e d u c t i o n of the C - l l keto p o s i t i o n of 11-dehydroc o r t i c o s t e r o n e or c o r t i s o n e to the 118 only i n compounds having  hydroxy form  occurred  4 / \ the A -3keto r i n g s t r u c t u r e . (15).  They were unable to f i n d any r e d u c t i o n i n compounds a s a t u r a t e d r i n g A. 4  Since  A -3-keto s t e r o i d s t h i s was  t h i s r e d u c t i o n occurred  questioned  This s p e c i f i c i t y of  3oc,llB-hydroxy-5aH  s t e r o i d s i n the l i v e r but not t h e i r corresponding d e r i v a t i v e s suggesting  11-oxo  t h a t the r i n g A s a t u r a t e d compounds  have undergone r e d u c t i o n of t h e i r C - l l ketone grouping.  They presented 116  reduct-  by Bush and Mahesh (16) a f t e r t h e i r demon-  s t r a t i o n of the occurrence of three  may  only i n  p o s t u l a t e d as a p o s s i b l e s i t e  of enzyme substrate attachment. i o n was  having  the hypothesis  t h a t the p a t t e r n of 11-oxo  hydroxy s t e r o i d s found i n u r i n e might be explained  and  i f the  w e l l known o x i d a t i o n r e d u c t i o n of 11-oxygen f u n c t i o n s i n v i v o was  l i m i t e d to those s t e r o i d s having  surface over the A and B r i n g s .  a more or l e s s f l a t  T h i s appeared  a  reasonable  since r e d u c t i o n at C - l l i s s t e r e o s p e c i f i c and must presumably occur by a s s o c i a t i o n of the a side of the s t e r o i d with enzyme r e s p o n s i b l e f o r the r e a c t i o n .  S t e r o i d s with a  the buckled  or bent A/B  r i n g j u n c t i o n would not be l i k e l y to combine as  f i r m l y with such an enzyme which was  a l r e a d y known to a c t  4  r a p i d l y on the A -3-keto forms which have a roughly A/B  r i n g system.  the s u b s t r a t e system was  I t was  f u r t h e r suggested, t h e r e f o r e ,  s p e c i f i c i t y of the  that  ll(3-hydroxy-dehydrogenase  determined l a r g e l y by the s t e r i c p r o p e r t i e s of  s t e r o i d , p a r t i c u l a r l y the A/B exception  planar  o c c u r r i n g i n the dog  ring junction.  With  one  (17), r e d u c t i o n appears to  e n t i r e l y to the ll|3-hydroxy form and  the  the l l a - h y d r o x y  of compounds such as C o r t i s o l have been shown to be  be  epimers biologi-  c a l l y i n a c t i v e . (12). Since  i t appears l i k e l y t h a t the adrenal  11-oxoster-  oids are not themselves b i o l o g i c a l l y a c t i v e on the t i s s u e s a f f e c t e d by a d r e n o c o r t i c a l hormones but are a c t i v e when administered  s y s t e m i c a l l y only because of t h e i r r a p i d and com-  p l e t e r e d u c t i o n to the 11(3 a l c o h o l f u r t h e r s t u d i e s were c a r r i e d on and two The  p o s s i b i l i t i e s were suggested.  f i r s t theory which was  and William-Ashman ( l 8 ) was  supported by T a l a l a y  t h a t these hormones exerted  their  a c t i o n i n a manner s i m i l a r to the coenzymes: i n t h i s case by a c t i n g as an e s s e n t i a l c o f a c t o r i n a redox system. b a s i s of experiments with e s t r a d i o l and p l a c e n t a l  0  n  the  isocitric  dehydrogenase (19) they proposed that s i n c e most s t e r o i d dehydrogenases i n animal t i s s u e can use both NAD coenzyme, s t e r o i d hormones i n general may  and NADP as  exert t h e i r a c t i o n  by c a t a l y z i n g transhydrogenation from reduced NAD  to. NADP.  - 6 They have shown that t h i s c a t a l y s i s of transhydrogenation i s e v i d e n t l y the b a s i s of the dehydrogenase i n v i t r o by  s t i m u l a t i o n of p l a c e n t a l e s t r a d i o l t h a t was  isocitric  observed by  V i l l e e , and p o i n t out t h a t the same dual requirement f o r pyridine nucleotide dehydrogenase of r a t  e x i s t s f o r the s t e r o i d 118-hydroxyliver.  Other r e s u l t s obtained by Bush and Mahesh Glenn, S t a f f o r d (20), and theory: namely, that the  others favour an a l t e r n a t e  general  118-hydroxy s t e r o i d hormones, not  the r e l a t e d 11-ketones, are the a c t i v e form and reduction  (16),  that  oxidation  of the C - l l oxygen f u n c t i o n i s not d i r e c t l y con-  cerned i n t h e i r mode of a c t i o n .  Bush and Mahesh r e l a t e the  l a t t e r theory to t h e i r observations  that C - l l reduction  occurs jln v i v o only i n s t e r o i d s having the r e l a t i v e l y a surface of the  over r i n g s A and B.  Thus l i t t l e ,  11-oxo s t e r o i d s occurred  buckled A/B  i f any,  reduction  with those which have the  r i n g j u n c t i o n t y p i c a l of 5<S H s t e r o i d s ( l l - o x o -  aetiocholanone or t e t r a h y d r o c o r t i s o n e ) . 5«x epimers with f l a t A/B  On the  and  that C o r t i s o l  Studies  re-  allotetrahydrocortisone).  Metabolism of c o r t i c o s t e r o i d s has other t i s s u e s .  other hand,  r i n g j u n c t i o n s were e x t e n s i v e l y  duced (ll-oxoandrosterone  been examined i n  by Sweat and Bryson ( 2 l ) have shown  can be degraded i n muscle t i s s u e by at l e a s t  f i v e separate pathways, namely cleavage of the C-17 o x i d a t i o n of the C - l l hydroxyl, duction  flat  at C-4,5  and C-20.  side  deoxygenation a t C-17  I f the a c t i o n of C o r t i s o l  and  chain, re-  is via  - 7 a chemical process i t i s p o s s i b l e one or more of these r e a c t i o n s i s i n v o l v e d i n a key hormonal a c t i v a t i o n process. Cortisol  As  i s known to modify a number of p h y s i o l o g i c a l p r o -  cesses ( e l e c t r o l y t i c , g l y c o g e n i c ,  anti-inflammatory,  i t i s a l s o p o s s i b l e that the d i f f e r e n t chemical  etc. )  conversions  are a s s o c i a t e d with separate p h y s i o l o g i c a l processes.  The  work of Sweat and Bryson confirms the hypothesis that r e duction of c o r t i s o n e to C o r t i s o l , b e l i e v e d to be the a c t i v a t i o n , i s p r i m a r i l y a h e p a t i c f u n c t i o n (21).  In muscle  there does not seem to be t h i s marked tendency towards C - l l r e d u c t i o n compared to other r e a c t i o n s .  These  observations  are a l l c o n s i s t e n t with the r e c o g n i t i o n t h a t c o r t i s o n e administered  s y s t e m a t i c a l l y has e f f e c t s comparable with  s o l but when employed l o c a l l y i s much l e s s a c t i v e . example, i n l o c a l anti-inflammatory  Corti-  For  reactions C o r t i s o l  has  been estimated 7-8 times as e f f e c t i v e as c o r t i s o n e . (22). With the observation at C - l l i n l i v e r  of t h i s c h a r a c t e r i s t i c r e d u c t i o n  o x y s t e r o i d metabolism, s t u d i e s were i n s t i t u t e d  on the d i s t r i b u t i o n of enzyme systems r e s p o n s i b l e f o r the r e a c t i o n s at C - l l of the c o r t i c o s t e r o i d s w i t h respect to t h e i r l o c a l i z a t i o n i n the d i f f e r e n t t i s s u e s and s u b c e l l u l a r f r a c t i o n s . Hurlock and T a l a l a y (23) reported the l o c a l i z a t i o n i n mammalian  l i v e r microsomes of an 118-hydroxy s t e r o i d dehydrogenase  which c a t a l y z e s a f r e e l y r e v e r s i b l e i n t e r c o n v e r s i o n of Cortisol  and c o r t i s o n e .  with e i t h e r NAD  T h i s enzyme f u n c t i o n s e q u a l l y w e l l  or NADP as hydrogen c a r r i e r .  The same workers  -  8  -  a l s o determined the i n t r a c e l l u l a r  l o c a t i o n of the 116-  hydroxy-dehydrogenase i n r a t l i v e r fugation.  The m i t o c h o n d r i a and microsome f r e e  were i n a c t i v e .  occurred.  Addition  f o r r e d u c e d NAD,  o f the o x i d i z e d  the major organ bolism.  liver,  C - l l reduction  coenzyme r e s u l t e d  i n the  k i d n e y has b e e n c o n s i d e r e d t o be  responsible f o r Cortisol  a n d c o r t i s o n e meta-  In a s t u d y by Mahesh and U l r i c h (24) c o r t i s o n e and  C o r t i s o l were m e t a b o l i z e d  either not at a l l or only to a  extent i n the b r a i n ,  tract,  supernatant  reaction. Other t h a n  small  centri-  However, i f t h e microsomes were s u p p l e m e n t e d  w i t h a g e n e r a t i n g system  reverse  by d i f f e r e n t i a l  diaphragm, h e a r t ,  thymus and s k e l e t a l m u s c l e  the k i d n e y . appreciable  In p a r t i c u l a r ,  gastrointestinal  of the r a t as compared t o  o x i d a t i o n a t C - l l o c c u r r e d to an  e x t e n t o n l y i n t h e k i d n e y and a p p e a r e d  main m e t a b o l i c  t r a n s f o r m a t i o n of C o r t i s o l  t o be t h e  by r a t k i d n e y .  Re-  d u c t i o n a t C - l l i n c o r t i s o n e was much l e s s t h a n the o x i d a t i o n o f the r e d u c e d  compound i n d i c a t i n g  different  the 1 1 8 - h y d r o x y - d e h y d r o g e n a s e f o u n d  from  this  enzyme e q u i l i b r i u m i s  liver.  The k i d n e y  enzyme, l i k e t h e l i v e r  utilize  e i t h e r NAD  o r NADP.  cellular system, genate bolism.  fractions  o f sub-  the l o c a t i o n o f t h e enzyme  t h e n u c l e a r and m i c r o s o m a l were o b s e r v e d  enzyme, c o u l d  In the i n v e s t i g a t i o n  t o determine  in rat  fractions  of k i d n e y  to have the g r e a t e s t e f f e c t  homo-  on C - l l meta-  The o b s e r v a t i o n t h a t most of t h e enzyme a c t i v i t y i n  r a t k i d n e y was a s s o c i a t e d w i t h microsomes a n d n u c l e i appear t o c o r r e l a t e w i t h m o r p h o l o g i c a l fractions.  would  s t u d i e s of t h e two  Prom e l e c t r o n m i c r o s c o p i c s t u d i e s o f l i v e r a n d  p a n c r e a t i c microsomes Palade and Seikewitz  (25)  suggested  these p a r t i c l e s are d e r i v e d from the endoplasmic r e t i c u l u m . Since the membrane of the endoplasmic r e t i c u l u m i s  continuous,  at l e a s t i n t e r m i t t e n t l y , with the c e l l membrane and  nuclear  membranes t h i s can be p o s t u l a t e d as an e x p l a n a t i o n f o r the dual l o c a t i o n of enzyme a c t i v i t y a s s o c i a t e d here with metabolism of C o r t i s o l and c o r t i s o n e .  In a p r e p a r a t i o n of i s o -  l a t e d n u c l e i by Dounce's method no o x i d a t i o n or r e d u c t i o n at C - l l could be demonstrated. p r e v i o u s l y observed  This i n d i c a t e d that the  activity  a s s o c i a t e d with a p r e p a r a t i o n of n u c l e i  contaminated with microsomes must be l o c a l i z e d i n the  micro-  somal p o r t i o n . (44). In an i n v i v o study on the metabolism of c o r t i c o sterone. and 11-dehydrocorticosterone served by Darrach  by the mouse i t was  et a l (26) t h a t intravenous  11-dehydrocorticosterone  resulted i n i t s rapid  and the r e l a t e d appearance of c o r t i c o s t e r o n e . curve f o r c o r t i c o s t e r o n e i n l i v e r was  ob-  i n j e c t i o n of disappearance The  decay  e s s e n t i a l l y the same  as found p r e v i o u s l y f o l l o w i n g a d m i n i s t r a t i o n of c o r t i c o s t e r o n e . The m e t a b o l i t e , 20a-rdihydrocorticosterone a l s o accumulates. No t r a c e s of C-20  reduced  11-dehydrocorticosterone  found i n blood or l i v e r a f t e r any studied.  Although  by t h i s C-20  c o u l d be  of the time i n t e r v a l s  the 11-keto form i s attacked more r e a d i l y  reductase  i n v i t r o i t i s apparent t h a t i n v i v o  r e d u c t i o n occurs very r a p i d l y at the C - l l ketone and p r i o r to r e d u c t i o n of the C-20  probably  group.  At present the 118-hydroxyl i s recognized as a major  - 10 f u n c t i o n a l group of adrenal a t i o n s a t C - l l represent  c o r t i c o s t e r o i d s (12) and a l t e r -  important metabolic changes.  The  i n t e r c o n v e r s i o n o f the 116-hydroxy and 11-keto forms i n v i v o has been observed i n numerous s t u d i e s based on a n a l y s i s of u r i n a r y metabolites  (27).  demonstrated i n perfused  T h i s r e a c t i o n has a l s o been  l i v e r of r a t (23) and dog (17), and  i n v i t r o i n r a t , beef, and p i g l i v e r  (28), r a t kidney (29),  bovine muscle (21), and i n f i b r o b l a s t s (30), lymphocytes and other t i s s u e s of s e v e r a l s p e c i e s .  The l i v e r i s probably the  main s i t e of r e d u c t i o n of 11-keto s t e r o i d s to the 118-hydroxyl d e r i v a t i v e s thus y i e l d i n g b i o l o g i c a l l y a c t i v e g l u c o c o r t i c o i d s . In extrahepatic  t i s s u e s , however, c o n d i t i o n s favour  dehydro-  genation o f the H B - h y d r o x y l to an 11-ketone group ( 3 l ) . P a r t i a l l y p u r i f i e d enzymes have been prepared from microsomes of r a t l i v e r  (23), kidney (24), and human l i v e r and p l a c e n t a  (32) which c a t a l y z e the r e v e r s i b l e r e d u c t i o n and dehydrogenation of the C - l l oxygen of the v a r i o u s c o r t i c o s t e r o i d s and which u t i l i z e p y r i d i n e n u c l e o t i d e s study the author wishes t o present and  as c o f a c t o r s .  In t h i s  s t u d i e s on the i s o l a t i o n  c h a r a c t e r i z a t i o n of the mouse l i v e r enzyme r e s p o n s i b l e  f o r the i n v i v o observation  ( l ) that the f i r s t  metabolism by the mouse of i n t r a v e n o u s l y  step i n the  i n j e c t e d 11-dehydro-  c o r t i c o s t e r o n e i s r e d u c t i o n at the C - l l p o s i t i o n to the 116hydroxyl  f u n c t i o n - the b i o l o g i c a l l y a c t i v e form (12).  E X P E R I M E N T A L  Preparation  of mouse l i v e r f r a c t i o n s .  One parts  p a r t f r e s h mouse l i v e r was  of c o l d 0.25  homogenized w i t h 4  M sucrose i n a g l a s s t i s s u e g r i n d e r  w i t h a t e f l o n p e s t l e having .006  - .009  fitted  inch clearance.  G i n an  p r e c i p i t a t e a f t e r c e n t r i f u g a t i o n f o r 20 minutes at 600 I n t e r n a t i o n a l r e f r i g e r a t e d c e n t r i f u g e was the from  supernatant s o l u t i o n S^.  Fractions  The  designated P^  and  and S£ were obtained  a f t e r c e n t r i f u g a t i o n f o r 15 minutes at 6,000 G i n a  S e r v a l l high speed c e n t r i f u g e and  f r a c t i o n s P^  and  from  S£ a f t e r c e n t r i f u g a t i o n f o r 60 minutes at 100,000 G i n the Spinco Model E u l t r a c e n t r i f u g e . (34). phosphate b u f f e r  (33) and  One  5 p a r t s of 0.25  p a r t Krebs Ringer M sucrose were added  to each p r e c i p i t a t e to b r i n g the volume up to t h a t before centrifugation.  Since no l o s s of enzyme a c t i v i t y occurred  storage at -10°C  of f r o z e n P^ preparations  made at one  on  l a r g e batches were  time, f r o z e n i n 25 ml q u a n t i t i e s and  thawed as  r e q u i r e d f o r each experiment.  Incubation and The contained  e x t r a c t i o n of mouse l i v e r f r a c t i o n s . i n c u b a t i o n medium, unless  2 mis  of a t i s s u e p r e p a r a t i o n  above; 65 mg niacinamide, 15 mg nicotinamide-adenine-dinucleotide (NADPH ), or 4 mg*  i n d i c a t e d otherwise, prepared as  sodium fumarate and  outlined 3 mg.  phosphate, reduced form  of the o x i d i z e d form (NADP), when the  of '  - 12 -  o x i d a t i v e r e a c t i o n was  being s t u d i e d , each added i n 2 ml  Krebs Ringer phosphate b u f f e r at pH 7.4. s u b s t r a t e was  added i n 0.2  a d j u s t e d to 10 ml.  (35).  The  steroid  ml ethanol and the t o t a l volume  The mixtures were incubated under  at  38°C f o r 2 hours i n a Dubnoff incubator shaking at 50 c y c l e s per minute.  The i n c u b a t i o n mixture was  25 ml chloroform which was  e x t r a c t e d with 5 x  washed w i t h 10 mis a l k a l i , 10  mis  a c i d and 10 mis water, d r i e d over anhydrous sodium sulphate, f i l t e r e d through  s i n t e r e d g l a s s and evaporated  i n vacuo at  40°C.  Chromatography and I s o l a t i o n of Compounds. The r e s i d u e a f t e r evaporation was on methanol washed (5X) Whatman No. formamide system. (36).  chromatographed  1 paper i n a benzene  Chromatograms were run 7-8  to give good s e p a r a t i o n of c o r t i c o s t e r o n e and costerone, the l a t t e r moving approximately that  hours  11-dehydrocorti-  twice as f a r i n  time. The  zones were detected by u l t r a v i o l e t  contact  photographs, the spots cut out and s t e r o i d e l u t e d w i t h methanolchloroform ( l : l ) . and d r i e d under N£.  E l u a t e s were f i l t e r e d through  sintered glass  I s o l a t e d compounds were then measured  q u a n t i t a t i v e l y by the blue t e t r a z o l i u m method. (37).  This  c o l o r i m e t r i c determination i s s p e c i f i c f o r s t e r o i d s with i n t a c t a-ketol side chain.  The  an  reducing p r o p e r t i e s of t h i s  s i d e chain are r e s p o n s i b l e f o r p r o d u c t i o n of the coloured  - 12a formazan complex which gives r i s e to a s p e c i f i c  absorption  peak at 520 m\i. Pooled samples of the products  i n each case were a l s o  c h a r a c t e r i z e d by mixed chromatograms with authentic compounds. S u l p h u r i c a c i d chromogens  (38) were obtained from c o l l e c t e d  samples and the a b s o r p t i o n s p e c t r a compared with a u t h e n t i c standards.  R E S U L T S I s o l a t i o n of 116 reductase  activity.  The present study was  f i r s t d i r e c t e d , towards l o c a l -  i z a t i o n i n a f r a c t i o n of homogenized mouse l i v e r of the reductase a c t i v i t y .  Table 1 records the a c t i v i t y of v a r i o u s  f r a c t i o n s of mouse l i v e r i n reducing to c o r t i c o s t e r o n e . A c t i v i t y was  11-dehydrocorticosterone  found c o n s i s t e n t l y to be  absent from the supernatant f o l l o w i n g h i g h speed and was  116  recovered i n the p r e c i p i t a t e  centrifugation  to the extent of  approximately h a l f t h a t found i n whole homogenate (WH) supernatants a f t e r c e n t r i f u g a t i o n a t lower speeds. and r e c e n t r i f u g a t i o n d i d not a l t e r the a c t i v i t y . t r a c e s of presumably C-20  or  Washing Faint  reduced products were observed  on  the chromatograms but i n amounts too small to c h a r a c t e r i z e . When chromatograms were s t a i n e d w i t h blue t e t r a z o l i u m no evidence of r i n g A reduced metabolites could be observed i n the P^ f r a c t i o n .  In the supernatant f r a c t i o n both C-20  duced and r i n g A reduced metabolites were obtained  re-  although  a f i v e f o l d i n c r e a s e i n coenzyme c o n c e n t r a t i o n was r e q u i r e d to demonstrate the l a t t e r .  In P^,  even w i t h the higher co-  enzyme l e v e l s , no r i n g A r e d u c t i o n was  demonstrable.  The  s u b s t r a t e and product could be recovered from chromatograms q u a n t i t a t i v e l y w i t h i n experimental e r r o r .  For example, i n  an i n c u b a t i o n of 250 ug of s t e r o i d approximately 75-85$ could be recovered i n product and s u b s t r a t e .  T h i s agrees w e l l  with the recovery values r e p o r t e d i n the l i t e r a t u r e .  (39).  - 14 Hence i t appears  t h a t f r a c t i o n P^ does not c o n t a i n s i g n i -  f i c a n t amounts of other enzymes a t t a c k i n g e i t h e r  cortico-  sterone or 11-dehydrocorticosterone.  R e v e r s i b i l i t y of the 116 reductase system:  116 hydroxyde-  hydrogenase a c t i v i t y of P^ I t was shown (Table 11) that when the P^ prepara t i o n was incubated with NADP as coenzyme and fumarate omitted, dehydrogenation  of c o r t i c o s t e r o n e t o 11-dehydro-  c o r t i c o s t e r o n e occurred, and i n much g r e a t e r y i e l d than the reduction reaction.  Substrate s p e c i f i c i t y of f r a c t i o n P^: As shown i n Table 11 the enzyme p r e p a r a t i o n i s capable of e f f e c t i n g the r e d u c t i o n of s e v e r a l s u b s t r a t e s : c o r t i s o n e , 11-dehydrocorticosterone, and 20a dihydro-11dehydrocorticosterone.  The dehydrogenation  of C o r t i s o l and  c o r t i c o s t e r o n e could a l s o be demonstrated with the P^ enzyme.  Coenzyme dependence of crude 116 reductase of mouse l i v e r : No measurable amount of 116 reductase a c t i v i t y i n f r a c t i o n P^ was observed While  i n the absence of added coenzyme.  a d d i t i o n of NAD or NAD^ r e s u l t e d i n p r o d u c t i o n of a  small amount of product the y i e l d was 8 times g r e a t e r w i t h NADP or NADPH^ i n d i c a t i n g a g r e a t e r degree of s p e c i f i c i t y  - 15 TABLE I 11-DEHYDROCORTICOSTERONE-118-REDUCTASE ACTIVITY IN MOUSE LIVER FRACTIONS  FRACTION  CORTICOSTERONE FORMED, Ug MEAN  TO  l  S  S  2  S  3  F  3  Conditions:  122 160 138 0 71  5 ml t i s s u e p r e p a r a t i o n + 15 mg sodium fumarate + 65 mg niacinamide + 2 mg NADB^  + 2 mg NADPH2 i n a t o t a l o f 6 ml Krebs-Ringer  Phos-  phate b u f f e r , pH 7.4; 1 mg 11-dehydrocorticosterone i n 0.2 ml ethanol added; incubated under ^ f o r 2 hours.  a t 38°C  N e u t r a l chloroform e x t r a c t of i n c u b a t i o n  mixture was chromatographed, measured  spectrophoto-  m e t r i c a l l y , by the U.V. and B-T methods and c h a r a c t e r ized.  -  16 -  f o r t h i s coenzyme i n both the o x i d i z i n g and reducing r e actions.  I t was i n s u r e d that the coenzyme was not a l i m i t -  i n g f a c t o r i n the i n c u b a t i o n mixtures by experiments i n which the coenzyme l e v e l was i n c r e a s e d w i t h no increase i n the amount of product formed.  K i n e t i c s of the 118 reductase system: A.  116 reductase a c t i v i t y of P^: 1.  E f f e c t of enzyme c o n c e n t r a t i o n  on r e a c t i o n v e l o c i t y .  F i g . 1 shows t h a t the r e a c t i o n v e l o c i t y i s p r o p o r t i o n a l to enzyme c o n c e n t r a t i o n  e_ over the range  £ = 0 to £ = 1.0 where e_ = 1.0 i s the a r b i t r a r y value  of e. assigned  to t h a t of the standard r e -  a c t i o n mixture.  2.  E f f e c t of time on the y i e l d of product. The  conversion  o f 11-dehydrocorticosterone to  c o r t i c o s t e r o n e i s p r o p o r t i o n a l to time f o r at l e a s t 2-1/2 hrs as shown i n F i g . I I . Thus the y i e l d of product a f t e r the a r b i t r a r y i n c u b a t i o n p e r i o d of 2 hours, used i n standard serves as a b a s i s f o r expressing v.  conditions,  reaction velocity  TABLE I I REDUCTION AND DEHYDROGENATION AT C - l l OF VARIOUS CORTICOSTEROIDS BY P  116  0  FRACTION OF MOUSE LIVER  Reductase A c t i v i t y with NADPH^ and Fumarate  Substrate  Product  Y i e l d , ug  corticosterone  71  20a-dihydro-ll-dehydro-  20a-dihydro-  30  corticosterone  corticosterone  11-dehydrocorticosterone  cortisone  Cortisol  49  116-HYDROXY DEHYDROGENASE ACTIVITY YflTH NADP Substrate Product Y i e l d , ug Corticosterone  11-dehydro-  347  corticosterone Cortisol Conditions:  Cortisone  339  2 ml enzyme p r e p a r a t i o n + 500 ug s t e r o i d as i n d i c a t e d i n 0.2 ml ethanol + 65 mg niacinamide  + 2 mg NADP or 2 mg NADP^ + 15 mg sodium fumarate, as i n d i c a t e d , i n 6 ml Krebs-Ringer phosphate b u f f e r , pH 7.4; i n cubated under N^ a t 38°C f o r 2 hours.  Neutral  chloroform  e x t r a c t s of i n c u b a t i o n mixture were chromatographed on paper and  zones e l u t e d and measured  spectrophotometrically.  - 18 -  3.  E f f e c t of s u b s t r a t e c o n c e n t r a t i o n on r e a c t i o n v e l o city. With the high coenzyme c o n c e n t r a t i o n o f the standard r e a c t i o n mixture constant  and s u b s t r a t e  concentra-  t i o n s_ v a r i a b l e over the range s = 14 x lO "* M to -  _5 s_ = 140 x 10  M, the e f f e c t of s_ on r e a c t i o n  v e l o c i t y v i s shown by the Dixon-Lineweaver-Burk plot  (40) i n P i g . I l l t h a t gives V  (imoles/ml/min and B»  = 1.6 x 1 0 "  4  = 1.8 x 10~^.  116-hydroxy-dehydrogenase a c t i v i t y of P^ 1.  E f f e c t of enzyme c o n c e n t r a t i o n of r e a c t i o n v e l o c i t y . F i g . IV shows t h a t r e a c t i o n v e l o c i t y f o r the dehydrogenation  i s p r o p o r t i o n a l to enzyme c o n c e n t r a t i o n  e_ over the range e = 0 to e = 1.0. where £ = 1.0 i s the a r b i t r a r y value of e_ assigned to t h a t of the standard  2.  r e a c t i o n mixtures.  E f f e c t of time on the y i e l d of product. The  conversion  costerone  of c o r t i c o s t e r o n e t o 11-dehydrocorti-  i s p r o p o r t i o n a l to time f o r at l e a s t  hours as shown i n F i g . V.  2-1/2  Thus the y i e l d of product  a f t e r the a r b i t r a r y i n c u b a t i o n p e r i o d of 2 hours used i n standard c o n d i t i o n s s e r v e s expressing  r e a c t i o n v e l o c i t y v.  as a b a s i s f o r  E f f e c t of s u b s t r a t e c o n c e n t r a t i o n on r e a c t i o n velocity. With an excess of coenzyme, the r e a c t i o n mixture constant and s u b s t r a t e c o n c e n t r a t i o n _s v a r i a b l e -4 over the range s = 1.4 x 10  -4 M t o s = 3.6 x 10  M  the e f f e c t of _s on r e a c t i o n v e l o c i t y v i s shown by the Dixon-Lineweaver-Burk p l o t i n F i g . VI which gives V = 2.7 x 10~ umoles/ml/min and K = max ^ m -4  -  l.0=C0NCENTRATI0N ENZYME  IN  20 -  STANDARD  INCUBATION  FIGURE I E f f e c t of enzyme c o n c e n t r a t i o n on r e a c t i o n v e l o c i t y , Standard c o n d i t i o n s (see t e x t ) over a range = 0 t o e_ = 1.0 where _e = 1.0 i s the a r b i t r a r y value of e_ signed to t h a t of the standard r e a c t i o n  mixture.  -  21 -  FIGURE I I E f f e c t of time on the y i e l d of c o r t i c o s t e r o n e . The ditions  enzyme was incubated under standard con-  (see t e x t ) over p e r i o d s up to 2-1/2 hours.  -  22  -  FIGURE I I I E f f e c t of s u b s t r a t e c o n c e n t r a t i o n on r e a c t i o n The ditions  enzyme was  velocity.  incubated under standard  con-  (see t e x t ) over a s u b s t r a t e c o n c e n t r a t i o n range of  s = 14 x 10" M 5  to s = 140 x 10" M. 5  V  = 1.6  x 10  K  = 1.8  x  10"  umoles/ml/min. 3  -  23  -  FIGURE I V  E f f e c t of enzyme c o n c e n t r a t i o n on r e a c t i o n v e l o c i t y . The enzyme was incubated under standard cond i t i o n s (see t e x t ) over a range of e = 0 to e -  1.0 where  £ = 1.0 i s the a r b i t r a r y value of e_ assigned t o t h a t of the standard r e a c t i o n mixture.  - 24 -  TIME  IN  MINUTES  AT  37° C  FIGURE V E f f e c t of time on the y i e l d of 11-dehydrocorticosterone The enzyme was incubated under standard ditions  (see t e x t ) over periods up to 2-1/2 hours.  con-  -  -  25  FIGURE V I E f f e c t of substrate c o n c e n t r a t i o n on r e a c t i o n v e l o c i t y The enzyme was ditions  incubated under standard  con-  (see t e x t ) over a s u b s t r a t e c o n c e n t r a t i o n range of  s = 1.4 x 10" M 4  to s = 3.6 x V  max  K  =  2  ,  10" M 4  7  x  1  0  = 1.7 x  umoles/ml/min. 10"  4  - 26 -  D I S C U S S I O N  I t was observed by Southcott e t a l ( l ) t h a t i n f u s e d 11-dehydrocorticosterone disappeared r a p i d l y  from  the blood of mice and a r e l a t e d appearance of c o r t i c o s t e r o n e occurred which reached maximum values a t 30 minutes post i n fusion.  No 11-dehydrocorticosterone c o u l d be detected i n  blood taken l a t e r than 5 minutes a f t e r i t s i n f u s i o n and a t no time c o u l d t h i s compound be found i n the l i v e r .  The  decay curve f o r c o r t i c o s t e r o n e i n l i v e r was e s s e n t i a l l y the same as found p r e v i o u s l y f o l l o w i n g a d m i n i s t r a t i o n of c o r t i costerone.  I t should be noted t h a t no t r a c e of 20a d i h y d r o -  11-dehydrocorticosterone was found i n e i t h e r blood or l i v e r at any of the time i n t e r v a l s s t u d i e d .  So, although the 11-  keto compound i s attacked by a crude C-20 reductase prep a r a t i o n from mouse l i v e r a t a much g r e a t e r r a t e than the 118 hydroxy compound, c o r t i c o s t e r o n e , i n v i t r o , i t i s apparent t h a t i n v i v o r e d u c t i o n occurs very r a p i d l y at the C - l l keto p o s i t i o n and p r i o r t o r e d u c t i o n of the C-20 keto group. (26). R e v e r s i b l e i n t e r c o n v e r s i o n s of hydroxy and keto s t e r o i d s have been demonstrated i n other  laboratories.  In a l l cases they are c a t a l y z e d by a c l a s s of w i d e l y d i s tributed, pyridine nucleotide-requiring hydroxy-steroid dehydrogenases which are s p e c i f i c w i t h r e s p e c t t o the p o s i t i o n and s t e r i c c o n f i g u r a t i o n o f the group reaction.  (23,24,27,17).  undergoing  - 27 The  P-j l i v e r f r a c t i o n used i n these e x p e r i -  ments was the p r e c i p i t a t e a f t e r c e n t r i f u g a t i o n a t 100,000 G f o r one hour of the supernatant from a 6,000 G c e n t r i f u g a t i o n f o r 20 minutes. mal  T h i s has been c a l l e d the microso-  f r a c t i o n (32) and t h i s i s the f r a c t i o n of mouse l i v e r  responsible  f o r i n t e r c o n v e r s i o n of 11-hydroxy and 11-keto  f u n c t i o n s of c o r t i c o s t e r o i d s . I s o l a t i o n of t h i s a c t i v i t y i n a microsomal f r a c t i o n i s i n agreement with the r e s u l t s of other workers. (23,24,32).  Hurlock and T a l a l a y r e p o r t the l o c a l i z a t i o n i n  mammalian and r a t l i v e r microsomes of an 118 h y d r o x y s t e r o i d dehydrogenase c a t a l y z i n g the r e v e r s i b l e i n t e r c o n v e r s i o n o f C o r t i s o l and c o r t i s o n e . bolism  Mahesh and U l r i c h s t u d i e d the meta-  of C o r t i s o l a t C - l l by r a t kidney and here again the  microsomal f r a c t i o n contained  a large p a r t of the a c t i v i t y .  A c o r t i c o s t e r o i d 118-hydroxy-dehydrogenase was i s o l a t e d from the microsomal f r a c t i o n of human l i v e r and p l a c e n t a by Meigs and Engel. The  mouse l i v e r 118 reductase shows a g r e a t e r  coenzyme s p e c i f i c i t y than these s i m i l a r microsomal enzymes i s o l a t e d from other organs and s p e c i e s .  In t h i s case NADP  and NADPH2 ^"kivi'ky a s shown to be 8 times t h a t of NAD w  and NADR^.  F o r the human enzyme NAD or NADP f u n c t i o n e d  approximately e q u a l l y . (32). The kidney enzyme could f u n c t i o n with e i t h e r c o f a c t o r . (24).  also  - 28 As shown i n Table 11, a l t h o u g h the enzyme i s able t o i n t e r c o n v e r t corticosterone brium l i e s  cortisone  reduced  as w e l l as  and 1 1 - d e h y d r o c o r t i c o s t e r o n e , t h e e q u i l i -  f a r on t h e s i d e o f t h e 11 o x i d i z e d  Thus t h i s , may be c o n s i d e r e d direction  and C o r t i s o l  a r e v e r s i b l e r e a c t i o n with the  o f t h e r e a c t i o n d e p e n d e n t on t h e c o n c e n t r a t i o n o f  o r o x i d i z e d NADP.  However, the c o m p l e t e  from l i v e r  of 11-dehydrocorticosterone  derivative  i s c o n s i s t e n t with the concept t h a t  cause o f i t s r e l a t i v e l y h i g h (40,41,42) y i e l d s reversible  o r i t s 20a d i h y d r o  concentration  reduced metabolites  absence  l i v e r , be-  o f r e d u c e d NADP  as p r i m a r y p r o d u c t s o f  coenzyme d e p e n d e n t d e h y d r o g e n a s e r e a c t i o n s .  extrahepatic ly  compound.  tissues  In  (21) where t h e coenzyme i s p r e d o m i n a n t -  i n the o x i d i z e d f o r m o x i d a t i o n a t the C - l l h y d r o x y l of  corticosterone  and C o r t i s o l h a s been d e m o n s t r a t e d t o o c c u r  very  T h i s would e x p l a i n the h i g h  to  rapidly.  11 8 h y d r o x y m e t a b o l i t e s  ratio  o f 11 k e t o  found i n a l l t i s s u e s other  than  liver. The enzyme p e r f o r m i n g t h i s  r e a c t i o n h a s been  named as a d e h y d r o g e n a s e r a t h e r t h a n a r e d u c t a s e b e c a u s e of the  r e a c t i o n e q u i l i b r i u m w h i c h i s i n the d i r e c t i o n of dehydro-  genation i n a l l t i s s u e s . required  L i v e r i s the o n l y  coenzyme p r e d o m i n a n t l y i n r e d u c e d The a b i l i t y  paration  specificity  form.  o f t h i s mouse l i v e r  t o reduce a v a r i e t y o f substrates  of s u b s t r a t e  t i s s u e w i t h the  enzyme p r e -  suggests a l a c k  s i m i l a r t o the microsomal  systems o f r a t k i d n e y and l i v e r ,  enzyme  and o f human l i v e r a n d  placenta.  I t remains to be e s t a b l i s h e d whether s i n g l e or  m u l t i p l e 11-dehydrogenase systems are i n v o l v e d . The  more s p e c i f i c dependence of the mouse  enzyme on NADP i s d i f f e r e n t from the s i m i l a r enzyme i n other t i s s u e s and species where NAD and NADP are almost e q u a l l y effective.  However, comparison of other p r o p e r t i e s of t h i s  116 reductase system shows them to be s i m i l a r t o the others mentioned:  a l l show some a c t i v i t y with NAD, a c t with more  than one s u b s t r a t e , are found i n microsomal preparations and are r e v e r s i b l e . This s e r i e s of k i n e t i c data presented above i s the f i r s t attempt a t k i n e t i c c h a r a c t e r i z a t i o n of a c o r t i c o s t e r o i d 118 hydroxy-dehydrogenase.  The r e s u l t s show t h a t  i n the presence of excess coenzyme e q u i l i b r i u m i s i n favour of the dehydrogenation r e a c t i o n .  The enzymatic nature of  the r e a c t i o n i n both the reducing  and o x i d i z i n g r e a c t i o n s  was e s t a b l i s h e d .  Requirement f o r a s p e c i f i c coenzyme could  be demonstrated. The  reaction v e l o c i t y i s proportional to  enzyme concentration hour p e r i o d s t u d i e d .  and l i n e a r with time over the 2-1/2 Substrate  a l s o determined enabling s  concentration  curves were  c a l c u l a t i o n of K and V „ f o r r e m max  duction of the C - l l ketone and o x i d a t i o n of the C - l l 6 hydroxyl function. I t i s g e n e r a l l y accepted t h a t the 116 hydroxyl i s the b i o l o g i c a l l y a c t i v e hormone (12) so i n t e r c o n v e r s i o n  - 30 of the 118 hydroxyl and 11 ketone i s of the utmost I n t e r c o n v e r s i o n occurs i n kidney (32),  fibroblasts  importance.  (29), muscle (21), p l a c e n t a  (30), and lymphocytes ( 4 3 ) . In a l l the  l a t t e r i n s t a n c e s c o n d i t i o n s favour dehydrogenation  and only  i n l i v e r i n v i v o does r e d u c t i o n appear favoured. E f f i c i e n t r e d u c t i o n by the l i v e r serves to maintain s t e r o i d i n the reduced c o n d i t i o n .  Other l i v e r enzymes  producing the b i o l o g i c a l l y i n a c t i v e p r o d u c t s - r i n g A reductases and s i d e bhain reductases- are more a c t i v e a g a i n s t 11 keto forms of the c o r t i c o s t e r o i d s than the l i p h y d r o x y l . (26). T h e r e f o r e , because l i p r e d u c t i o n seems t o occur f i r s t i n l i v e r , preceding other a l t e r a t i o n s , a p h y s i o l o g i c a l r o l e i s suggested.  T h i s can be i n t e r p r e t e d as a tendency  to econo-  mize on the d e s t r u c t i o n of s t e r o i d s by o f f s e t t i n g  inactiva-  t i o n by the other l i v e r enzymes and a l s o the dehydrogenation which occurs i n e x t r a h e p a t i c t i s s u e s .  - 31 S U M M A R Y The  i n v i v o observation  that the f i r s t  i n metabolism by the mouse of i n t r a v e n o u s l y dehydrocorticosterone  i s reduction  step  i n j e c t e d 11-  a t the C - l l p o s i t i o n t o  the l i p hydroxyl f u n c t i o n , l e a d to the present s t u d i e s . T h i s reductase a c t i v i t y was l o c a l i z e d i n the microsomal f r a c t i o n o f l i v e r which contains system c a t a l y z i n g the i n t e r c o n v e r s i o n l i p hydroxyl f u n c t i o n s  an enzyme  o f the 11 keto and  of corticosterone,  11-dehydrocorti-  costerone, 20a dihydro 11-dehydrocorticosterone, and  cortisone  Cortisol.  A r e l a t i v e s p e c i f i c i t y f o r NADP and NADPH as 2  cofactors  i n dehydrogenation and r e d u c t i o n  be demonstrated.  r e s p e c t i v e l y can  Each c a t a l y z e d 8 times the a c t i v i t y ob-  served with NAD o r NADH . 2  The  d i r e c t i o n o f the r e a c t i o n i s apparently  c o n t r o l l e d by the reduced or o x i d i z e d s t a t e of the coenzyme. C e r t a i n k i n e t i c p r o p e r t i e s o f the r e a c t i o n have been i n v e s t i g a t e d .  The r e a c t i o n v e l o c i t y i s p r o p o r t i o n a l  to enzyme c o n c e n t r a t i o n  and l i n e a r with time over the 2-1/2  hour p e r i o d s t u d i e d .  F o r the r e d u c t i o n  dehydrocorticosterone  a s substrate  K  m  r e a c t i o n with 11_5  = 1.8 x 10  and  —4 / / V = 1.6 x 10 umoles/ml/min. In the dehydrogenation -4 r e a c t i o n w i t h c o r t i c o s t e r o n e as substrate E = 1.7 x 10 m and V = 2.7 x 10 u.moles/ml/min.  - 32 B I B L I O G R A P H Y 1.  Darrach, M., In "Mechanisms of H y p e r s e n s i t i v i t y " ,  Little  Brown & Co., Boston, 613, (1959). 2.  C a s p i , E., Levy, H. and Hechter, 0., Arch. Biochem. and Biophy., 45, 169 (1953). L. and A x e l r o d , L., Metabolism, 3, 438 (1954).  3.  Miller,  4.  Hechter, 0., Solomon, M., Marchi, J . , C a s p i , E. and F e i n s t e i n , M., J . C l i n . E n d o c r i n o l , and Metabolism, 12, 935  5.  (1952).  I n g l e , D., Adrenal Cortex I I I , J o s i a h Macy J r . , Found. Conf., 107 (1951).  6.  Bush, L., and Mahesh, V., Biochem. J . , 71, 718 (1959).  7.  Wilson, H. , Fairbanks, R. , S c i a l a h b a , D., J . C l i n . crinoL and Metabolism, 14, 814 (1954).  8.  Hechter, 0., Frank, E., C a s p i , E . and Frank, H., Endo-  EndOrr  c r i n o l o g y , 60, 105 (1957). 9.  Schneider, J . , and Horstman, P., J . B i o l . Chem., 196, 629  (1952).  10.  Doughaday, W., P h y s i o l . Reviews, 39>  11.  Doughaday, ¥., J . C l i n . Invest., 37, 519 (1958).  12.  S a r r e t , L., Annals of the N. Y. Academy of Science, 82, 802 (1959).  13.  Forchelli, 443,  8 8  5  (1959).  I . , and Dorfman, R., J . B i o l . Chem., 223, (1956).  14.  U l r i c h , F., Biochem. J . , 61, 361 (1958).  15.  Hubener, H. J . , Fukushima, D. K., and G a l l a g h e r , F., J . B i o l . Chem., 220, 449 (1956).  16.  Bush, I . and Mahesh, V., Biochem. J . , 71, 705 (1959).  - 33 17.  A x e l r o d , L., and M i l l e r ,  L., Arch. Biochem. and Biophy.,  60, 373 (1956). 18.  T a l a l a y , P., and Williams-Ashman, Sci.  H.,  Proc. N a t l . Acad.  (Wash.), 44, 15 (1958).  19.  V i l l e e , C , J . B i o l . Chem., 215., 171 (1955).  20.  Glenn, E., S t a f f o r d , R., L i s t e r , S., and Bowman, B., Endocrinology, 61, 128 (1957).  21.  Sweat, M., and Bryson, M., Biochim.. e t Biophys. A c t a . , 44, 217 (1960).  22.  Dougherty,  L., Adrenal Cortex I I , J o s i a h Macy J r . , Pound.  Conf., 88 (1950). 23.  Hurloch, B. and 33alalay, P., Arch. Biochem. and Biophys., 80, 468 (1959).  24.  Mahesh, V., and U l r i c h ,  F., J . B i o l . Chem., 235, 356 ( i 9 6 0 ) .  25.  Palade, G., and S i e k e w i t z , P., J . B i o p h y s i c a l and B i o chemical Cytology, 2, 671 (1956).  26.  Darrach, M., K r e h b i e l , R. and Burton, A. F., unpublished work.  27.  Roberts, S., and Szego, E., Ann. Rev. Biochem, 24, 543 (1959).  28.  F i s h , A.,Hayano, N., and P i n c u s , G., A r c h . Biochem. and Biophys., 42, 480 (1953).  29.  Ganis, F., A x e l r o d , R., M i l l e r , 841  30.  L., J . B i o l . Chem., 218,  (1956).  Dougherty,  T., "The Leukemias",  Acad. P r e s s . , N.T.,  (1957). 31.  E n g e l , L., and Langer, L., Ann. Rev. Biochem., 30, 449 (1961).  32.  Meigs, R., and E n g e l , L., Endocrinology, (S9, 152 (1961).  - 34 33.  Krebs, H. A., and E g g l e s t o n , L. V., Biochem. J . , 34, 442  34.  (1940).  Hogeboom, G., and Schneider, W. C , In "The N u c l e i c A c i d s " , Vol.  I I , Chapt. 21, Ed. Chargaff, E., and Davidson,  J . N., N. I . , Acad. Press (1955). 35.  De Courcy, C , and Schneider, J . J . , J . B i o l . Chem., 223, 885 (1956).  36.  Z a f f a r o n i , A., and Burton, R. B., J . B i o l . Chem., 193, 749  37.  (1951).  Mader, W. J . , and Buck, R. R., A n a l y t i c a l Chemistry, 24, 666  (1952).  38.  Z a f f a r o n i , A., J . Am. Chem. S o c , 72, 3828 ( l 9 5 0 ) .  39.  Romanoff, L. P., M a r r i s , C. W., Welch, P., Rodriquez, R., and P i n c u s , G., J . C l i n . E n d o c r i n o l , and Metabolism, 21, 1413 (1961).  40.  Dixon, M., and Webb, E . C , "Enzymes," Longmans, London (1960).  41.  Bassham, J . A., B i r t , L. M., Hems, R., and Loening, V. E., Biochem. J . , 73, 491 (1959).  42.  S i l b e r , R., Gabrio, W. G., and Huennekens, F. M., J . C l i n . I n v e s t . , 41, 230 (1962).  43.  Dougherty, J . F., B e r l i n e r , M. L., and B e r l i n e r , D. L., Endocrinology, 66, 550 ( i 9 6 0 ) .  44.  Mahesh, V., and U l r i c h , £., Nature, 184, 1147 (1959).  1.0= CONCENTRATION  ENZYME  IN  STANDARD  INCUBATION  TIME  IN  MINUTES  AT  37° C  I.O=CONCENTRATION  ENZYME  IN  STANDARD  INCUBATION  I-  DEHYDROCORTICOSTERONE —  CORTICOSTERONE  

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