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An inhibitor of ornithine decarboxylase in the thymus and spleen of dexamethasone-treated rats Bishop, Paul Burton 1984

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AN INHIBITOR OF ORNITHINE DECARBOXYLASE IN THE THYMUS AND SPLEEN OF DEXAMETHASONE-TREATED RATS  by  PAUL BURTON BISHOP B-.Sc, THE UNIVERSITY OF BRITISH COLUMBIA VANCOUVER, B.C., 1974  THE  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE  FACULTY OF GRADUATE STUDIES DEPARTMENT OF BIOCHEMISTRY FACULTY OF MEDICINE UNIVERSITY OF BRITISH COLUMBIA  We a c c e p t t h i s t h e s i s as conforming _io, the r e q u i r e d s t a n d a r d  THE c)  UNIVERSITY OF BRITISH COLUMBIA . JULY 1984 PAUL BURTON BISHOP, 1984  In  presenting  requirements  this for  thesis  an  advanced  of  B r i t i s h Columbia,  it  freely  agree for  available  that  or  for  by h i s  understood  that  of  reference  and  study.  I  extensive  her  by  the  p u b l i c a t i o n of be  this  It  this  allowed without  Columbia  make  further  head  representatives.  not  The U n i v e r s i t y o f B r i t i s h 1956 Main M a l l Vancouver, Canada V6T 1Y3  copying of  granted  Biochemistry  August 7 , 1984  University shall  permission.  Department  the  Library  may b e  shall  at  the  the  for  or  f u l f i l m e n t of  that  copying or  financial gain  degree  agree  purposes  department  for  I  permission  scholarly  in partial  thesis  of  my  is thesis my  written  ii  ABSTRACT  The a c t i v i t y of o r n i t h i n e i n polyamine  d e c a r b o x y l a s e , the r a t e - d e t e r m i n i n g enzyme  b i o s y n t h e s i s , decreases markedly  i n r a t thymus and s p l e e n  soon a f t e r the i n j e c t i o n of r a t s w i t h dexamethasone. that as e a r l y as 2 1/2  hours a f t e r hormone treatment, when enzyme a c t i v i t y  i s v e r y low, an i n h i b i t o r of ODC readily  i s present.  Inhibition  d e t e c t e d a t 5 and 12 hours, but not a t 24 hours  glucocorticoid a c t i v i t y was  injection.  The  differentiates  s m a l l m o l e c u l a r weight  of ODC  It retains  i t from o t h e r ODC  can be  after  i n h i b i t o r appears to be a p r o t e i n  d e s t r o y e d by heat or t r y p s i n .  d i a l y s i s which  since i t s  i t s a c t i v i t y after  inhibitors  that  require  substances f o r a c t i v i t y .  The apparent m o l e c u l a r weight 54,000, which d i f f e r e n t i a t e s ODC  T h i s study shows  of a p a r t i a l l y p u r i f i e d  extract  was  t h i s i n h i b i t o r from antizyme, an i n h i b i t o r of  found i n s e v e r a l o t h e r c e l l  types.  The  i n h i b i t o r appears t o a c t by a  non-competitive and n o n - c a t a l y t i c mechanism s i n c e the amount of i n h i b i t i o n does not change w i t h time and i t s i n t e r a c t i o n enzyme's a f f i n i t y early  for ornithine.  event i n lymphoid  tissues  w i t h ODC  does not a f f e c t  The f o r m a t i o n of t h i s i n h i b i t o r i s an i n response t o dexamethasone and may  important i n c a u s i n g the i n h i b i t i o n of c e l l d i v i s i o n which precedes d e s t r u c t i o n of  lymphocytes.  be the  the  iii  TABLE OF CONTENTS Page Abstract List  of Tables  List  of F i g u r e s  iv v  L i s t of Abbreviations  vi  Acknowledgements  v i  1  Introduction  1.  General  1-  Polyamine B i o s y n t h e s i s  1.  The R e g u l a t i o n  4.  o f ODC A c t i v i t y  Polyamine Metabolism i n Thymus and Other Lymphatic T i s s u e  . . .  6.  The E f f e c t o f G l u c o c o r t i c o i d s on Polyamine Metabolism i n Lymphatic T i s s u e The Present  8.  Investigation  9.  M a t e r i a l s and Methods  11.  Results Conditions  19. 19.  f o r Measurement o f ODC A c t i v i t y  The Occurrence of an I n h i b i t o r o f ODC i n Lymphatic from Dexamethasone-Treated Rats  Tissue 25.  C h a r a c t e r i z a t i o n o f ODC I n h i b i t i n g Substance i n Thymus o f Dexamethasone-Treated  Rats  35.  P u r i f i c a t i o n of ODC I n h i b i t o r The R o l e o f P y r i d o x a l Phosphate  37. i n Thymus ODC I n h i b i t i o n . . . .  45.  Discussion  48.  Bibliography  52.  iv  LIST OF TABLES  TABLE  1.  2.  3.  4.  5.  6.  7.  8.  9.  10.  11.  Page  ODC A c t i v i t y i n Thymus E x t r a c t s Prepared U s i n g D i f f e r e n t Buffers  20.  ODC A c t i v i t y i n Thymus E x t r a c t s o f C o n t r o l Rats Using D i f f e r e n t D i t h i o t h r e i t o l Concentrations  21.  Determined  I n h i b i t i o n of ODC A c t i v i t y by Aqueous E x t r a c t s o f Thymus and S p l e e n from Dexamethasone-Treated Rats Occurrence of an I n h i b i t o r of Thymus and Spleen ODC i n L u b r o l E x t r a c t e d Lymphoid T i s s u e from Dexamethasone-Treated Rats  26.  .  28.  I n h i b i t i o n o f ODC i n Thymus and Spleen o f DexamethasoneT r e a t e d Rats as a F u n c t i o n o f I n c u b a t i o n Time  31.  A c t i v i t y o f Thymus ODC I n h i b i t o r a t D i f f e r e n t S u b s t r a t e Concentrations  32.  A c t i v i t y o f ODC and ODC I n h i b i t o r i n Thymus E x t r a c t s Prepared from Rats a t D i f f e r e n t Times a f t e r Dexamethasone Treatment . . .  34.  E f f e c t s of V a r y i n g t h e R a t i o o f I n h i b i t o r t o Enzyme on ODC Activity  36.  E f f e c t o f S m a l l M o l e c u l a r Weight Inhibitory Activity  39.  Substances on Thymus ODC . . . . . . . . . . .  P u r i f i c a t i o n o f Thymus ODC I n h i b i t o r u s i n g DEAE CL-6B Column Chromatography  Sepharose  P u r i f i c a t i o n of Thymus ODC I n h i b i t o r u s i n g H e p a r i n Sepharose CL-6B Column Chromatography  42.  44.  LIST OF FIGURES  FIGURE  Page  1.  L-Ornithine Kinetics  o f ODC i n Thymus o f C o n t r o l Rats  2.  E f f e c t o f I n c u b a t i o n Time on Thymus ODC A c t i v i t y  23.  3.  Effect  24.  4.  The E f f e c t o f L u b r o l on ODC A c t i v i t y i n Rat Thymus and Spleen  5.  L-Ornithine Kinetics  6.  E f f e c t o f T r y p s i n on A c t i v i t y o f Thymus ODC I n h i b i t o r  38.  7.  E f f e c t o f Sodium C h l o r i d e on Thymus ODC A c t i v i t y  41.  8.  Molecular  of Protein Concentration  22.  on Thymus ODC A c t i v i t y . .  o f Thymus ODC i n a M i x i n g Assay  Weight D e t e r m i n a t i o n  of Thymus ODC I n h i b i t o r  29. 33.  . . . . .  46.  vi  LIST OF ABBREVIATIONS  A  280  Absorbance measured a t 280  nm.  Hepes  N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic  Hepps  N-2-Hydroxyethylpiperazine-N'-3-propanesulfonic  Km  Michaelis-Menten  mCi  milliCurie  mM  millimolar  Mops  Morpholinopropanesulfonic acid  Mr  M o l e c u l a r weight  nmole  nanomole  ODC  ornithine  ODIF  o r n i t h i n e decarboxylase i n a c t i v a t i n g  pmole  picomole  rpm  r e v o l u t i o n s per minute  SAMD  S-adenosyl-L-methionine  SDS  Sodium d o d e c y l  UM  micromolar  PLP  p y r i d o x a l phosphate  BSA  Bovine Serum Albumin  constant  decarboxylase  sulfate  decarboxylase  factor  acid acid  vii  ACKNOWLEDGEMENTS  The work i n t h i s t h e s i s was performed under the s u p e r v i s i o n o f Dr. J.F. R i c h a r d s . appreciated.  His valuable  a s s i s t a n c e and a d v i c e were very much  I would a l s o l i k e t o thank D r s . D. Vance and M. Mauk f o r  t h e i r s u g g e s t i o n s and Mr. Ted Peng f o r h i s t e c h n i c a l a s s i s t a n c e .  The  f i n a n c i a l support o f the Canadian Memorial C h i r o p r a c t i c C o l l e g e i s gratefully  acknowledged.  1.  INTRODUCTION  General Polyamines are n a t u r a l c o n s t i t u e n t s c e l l s which are  incapable  p r o t e i n , l i p i d and s y n t h e s i s and  of s y n t h e s i z i n g  function (1).  The  intercellular a cell  changes i t s m e t a b o l i c f u n c t i o n ( 2 ) , and polyamine b i o s y n t h e t i c enzymes has wide v a r i e t y of systems w e l l b e f o r e  in  polynucleotide  concentrations  of these  d i v i d e s , d i f f e r e n t i a t e s or  the a c t i v i t y of one  or more of  the  been shown to i n c r e a s e markedly i n a a p r o l i f e r a t i v e response occurs  I f , as i s w i d e l y h e l d , polyamine metabolism i s i n e x t r i c a b l y  i n v o l v e d w i t h the c e l l u l a r growth p r o c e s s , biosynthesis  should  then decreased polyamine  be a s s o c i a t e d w i t h a c y t o l y t i c response and  mechanism of p r o d u c i n g t h i s response would be regulating c e l l  division.  A negative  decrease i n the a c t i v i t y of one demonstrated i n r a t thymus. of 400  and  them e x h i b i t marked d e f e c t s  c a r b o h y d r a t e metabolism, as w e l l as i n  compounds i s known to a l t e r b e f o r e  (3-7).  of most l i v i n g organisms,  fundamentally important i n  growth response preceded by a marked  polyamine b i o s y n t h e t i c enzyme has  The  enzyme a c t i v i t y was  response i s unknown. increase synthesis  Polyamine  The  I t may  i n degradation,  mechanism i n v o l v e d  been  reduced by a f a c t o r  at l e a s t 12 hours p r i o r to thymus i n v o l u t i o n and  lymphocyte c y t o l y s i s ( 8 ) .  the  eventual  i n mediating  this  i n v o l v e a d e c r e a s e i n enzyme s y n t h e s i s ,  a p o s t - t r a n s l a t i o n a l m o d i f i c a t i o n or  an  the  of an i n h i b i t o r t h a t a c t s s p e c i f i c a l l y on the enzyme.  Biosynthesis  Polyamines a r e m e t a b o l i c d e r i v a t i v e s of the amino a c i d s L-methionine and  L-ornithine.  The  t h r e e most common p r o d u c t s of the pathway are  the  2.  diamine  p u t r e s c i n e and the polyamines  decarboxylase  spermidine and spermine.  Ornithine  (ODC), which c a t a l y z e s the f o r m a t i o n of p u t r e s c i n e from  o r n i t h i n e , i s c o n s i d e r e d t o be the r a t e l i m i t i n g enzyme i n the b i o s y n t h e s i s o f polyamines  (9).  In r e s t i n g c e l l s ,  t h i s enzyme has the  lowest a c t i v i t y o f a l l the b i o s y n t h e t i c enzymes i n the pathway.  Iti s  w e l l c h a r a c t e r i z e d i n s e v e r a l t i s s u e s (10-14) and has been p u r i f i e d t o e l e c t r o p h o r e t i c homogeneity from a v a r i e t y o f s o u r c e s (15-17). a s t r o n g dependence on t h i o l s f o r a c t i v i t y and i s o f t e n found (18,19).  E s t i m a t e s o f m o l e c u l a r weight  ODC shows i n dimers  f o r t h e monomer range from 50,000  t o 55,000 i n mammalian systems (20-23) and as h i g h as 80,000 i n o t h e r systems (24,25). (26).  The enzyme r e q u i r e s p y r i d o x a l phosphate as a c o f a c t o r  M u l t i p l e s p e c i e s of ODC have been demonstrated  their a f f i n i t i e s  f o r p y r i d o x a l phosphate ( 2 7 ) .  which d i f f e r i n  These may r e p r e s e n t  p o s t - t r a n s l a t i o n a l m o d i f i c a t i o n s r a t h e r than t r u e isozymes  since there i s  no evidence t o support t h e e x i s t e n c e o f d i f f e r e n t ODC genes ( 2 8 ) . an a c i d i c p r o t e i n h a v i n g an i s o e l e c t r i c p o i n t i n the range 4.8 ( 2 9 ) .  ODC i s  of pH 4.1 t o  A f t e r p u r i f i c a t i o n , mammalian ODC has an optimum pH of  a p p r o x i m a t e l y 7.4 ( 3 0 ) . In e u k a r y o t e s , the enzyme has a r e p o r t e d range o f Km v a l u e s f o r o r n i t h i n e o f 0.03 t o 0.2 mM enzyme and has two important  (31).  ODC i s mainly a c y t o s o l i c  c h a r a c t e r i s t i c s t h a t d i s t i n g u i s h i t from  other known e u k a r y o t i c enzymes ( 3 2 ) .  One i s i t s i n d u c i b i l i t y .  The ODC  a c t i v i t y i n r e s t i n g a d u l t mammalian t i s s u e s , w i t h few e x c e p t i o n s , i s extremely low (33-35).  I n c r e a s e s i n a c t i v i t y o f up t o 1000 f o l d w i t h i n a  few hours have been r e p o r t e d i n response (36).  A second unique  t o a p o s i t i v e growth s t i m u l u s  c h a r a c t e r i s t i c o f ODC i s i t s v e r y s h o r t h a l f - l i f e .  Depending upon t h e type o f c e l l and stage o f d i v i s i o n , ODC can be l e s s than 20 minutes,  the h a l f - l i f e of  which I s much l e s s than t h a t o f any o t h e r  known e u k a r y o t i c enzyme.  Another  d e c a r b o x y l a s e , S-adenosyl-L-methionine  d e c a r b o x y l a s e (SAMD o  AdoMet DC) i s n e c e s s a r y f o r the s y n t h e s i s o f t h e h i g h e r spermidine and spermine.  polyamines  SAMD c o n v e r t s S-adenosyl-L-methionine  S-adenosyl-methyl-thiopropylamine  which a c t s as an aminopropyl  to . donor f o r  t h i s t r a n s f e r r e a c t i o n w i t h p u t r e s c i n e , t h a t o c c u r s l a t e r i n the pathway. While SAMD a l s o has a s h o r t h a l f - l i f e , different  i t s p r o p e r t i e s a r e fundamentally  from those o f ODC ( 3 7 ) . I t does n o t r e q u i r e p y r i d o x a l  as a c o f a c t o r , but i n s t e a d appears  phosphat  t o belong t o a s m a l l c l a s s o f amino  a c i d d e c a r b o x y l a s e s t h a t uses c o v a l e n t l y l i n k e d p y r u v a t e as a p r o s t h e t i c group.  I t s presence i s r e q u i r e d f o r enzyme  The  two remaining polyamine  transferases.  activity.  b i o s y n t h e t i c enzymes a r e propylamine  T h e i r p r o p e r t i e s c o n t r a s t w i t h the d e c a r b o x y l a s e s i n t h a t  both a r e s t a b l e enzymes w i t h l o n g h a l f - l i v e s called  spermidine and spermine  ( 3 8 ) . The t r a n s f e r a s e s ( a l s  synthase) do not r e q u i r e c o f a c t o r s and hav  c o n s i d e r a b l y h i g h e r a c t i v i t i e s than ODC and SAMD i n the r e s t i n g T h e i r a c t i o n i s t o t r a n s f e r an aminopropyl  group  cell.  t o p u t r e s c i n e and  s p e r m i d i n e which r e s u l t s i n t h e s y n t h e s i s o f spermidine and spermine respectively.  While  the a c t i v i t i e s  of the l a t t e r three b i o s y n t h e t i c  enzymes a r e o f t e n l i n k e d t o ODC a c t i v i t y , regulated independently.  i t appears  t h a t they can a l s o b  Large i n c r e a s e s i n ODC i n many s t i m u l a t e d  t i s s u e s can o c c u r i n d e p e n d e n t l y o f changes i n SAMD a c t i v i t y little  (39). Again,  i s known of t h e mechanisms f o r c o n t r o l l i n g the a c t i v i t i e s o f  spermidine and spermine of spermine  synthase.  Putrescine i s a competitive i n h i b i t o r  synthase and t h e r e f o r e h i g h c e l l u l a r  l e v e l s of putrescine  4.  r e s u l t i n g from a c t i v e growth f a v o r spermidine  The  Regulation  of ODC  S i n c e ODC  production.  Activity  i s the r a t e d e t e r m i n i n g  enzyme i n the b i o s y n t h e s i s  polyamines, the r e g u l a t i o n of polyamine metabolism i s c e n t e r e d modulating the a c t i v i t y of t h i s enzyme. the two  unique c h a r a c t e r i s t i c s of ODC,  When looked  p e r i o d of time.  can r i s e  i t s i n d u c i b i l i t y with  s h a r p l y , but  ODC  The  r e g u l a t i o n of ODC  (41).  ODC  been shown t o i n v o l v e changes i n the h a l f - l i f e of  the  C e l l s induced  i n ODC  increases  i n ODC  through  been demonstrated i n mouse f i b r o b l a s t s  a c t i v i t y i n transformed f i b r o b l a s t s  has  i n the amount of enzyme p r o t e i n ( 4 2 ) .  and  r a p i d d e a c t i v a t i o n of ODC concentrations  increased  r a t e s of ODC  a c t i v i t y has  A  a l s o been shown to r e s u l t  i n d i r e c t however, s i n c e a macromolecular ODC  been c a l l e d  with  synthesis.  of the polyamines themselves.  by micromolar c o n c e n t r a t i o n s I t has  Regulation  a c t i v i t y were shown to be a s s o c i a t e d  decreased r a t e s of d e g r a d a t i o n  (43).  (40).  been demonstrated i n androgen s t i m u l a t e d mouse k i d n e y ,  where l a r g e i n c r e a s e s  induced  the  w i t h glutamine show a f o u r f o l d i n c r e a s e i n  p r o t e i n s y n t h e s i s has  s i m i l a r e f f e c t has  o f t e n probably  short  ODC  been c o r r e l a t e d w i t h  from e l e v a t e d  the  In HeLa c e l l s ,  A 20 f o l d i n c r e a s e  The  or  a c t i v i t y appears to take  h a l f - l i f e when compared w i t h uninduced c e l l s  increased  of ODC,  o n l y f o r an extremely  p l a c e through a v a r i e t y of d i f f e r e n t mechanisms.  enzyme ( 4 0 ) .  a short  T h i s i m p l i e s t h a t a subsequent r a p i d d e a c t i v a t i o n of  enzyme must take p l a c e .  r e g u l a t i o n has  around  at i n combination,  h a l f - l i f e , mean t h a t the i n t e r c e l l u l a r c o n c e n t r a t i o n s enzyme's a c t i v i t y ,  of  of polyamines has  This action i s inhibitor  been demonstrated  'antizyme' as i t i s a p r o t e i n i n h i b i t o r of  an  5.  enzyme t h a t i s s y n t h e s i z e d  i n response t o the immediate or remote product  o f the r e a c t i o n i t c a t a l y z e s  ( 4 4 ) . Antizyme i s a s m a l l p r o t e i n of Mr  26,000 and was f i r s t  identified  w e l l as i n r a t l i v e r  (45).  upon c o n t i n u e d  i n L1210, neuroblastoma and H35 c e l l s as  The i n d u c t i o n o f t h i s i n h i b i t o r i s dependent  p r o t e i n s y n t h e s i s and i t s l e v e l s w i t h i n the c e l l have been  shown to d e c r e a s e r a p i d l y i n the presence o f cycloheximide  (46).  In r a t  l i v e r i t i s l o c a t e d i n the c e l l n u c l e u s and c o u l d be d i s s o c i a t e d from ODC i n v i t r o by h i g h s a l t c o n c e n t r a t i o n s K i n e t i c s t u d i e s have r e v e a l e d (48).  Substances having  been found i n o t h e r  r e s u l t i n g i n r e a c t i v a t e d ODC ( 4 7 ) .  t h a t i t i n h i b i t s ODC  non-competitively  c h a r a c t e r i s t i c s s i m i l a r t o those of antizyme have  t i s s u e s and as a r e s u l t  of d i f f e r e n t  stimuli.  A  p y r i d o x a l phosphate s e n s i t i v e ODC r e g u l a t o r y p r o t e i n of Mr 19,500 was induced  by p u t r e s c i n e  i n chicken  liver  (49).  The growth  stimulus  r e s u l t i n g from p l u c k i n g h a i r from the d o r s a l s k i n o f r a t s r e s u l t e d i n the production  o f an ODC i n h i b i t o r y substance o f apparent Mr g r e a t e r  30,000 ( 5 0 ) .  T h i s p r o t e i n has some a n t i z y m e - l i k e  than  p r o p e r t i e s and i s  thought t o be i n v o l v e d i n the c y c l i c a l r e g u l a t i o n o f h a i r growth i n r a t s .  ODC i s a l s o known t o be r e g u l a t e d modification.  through p o s t - t r a n s l a t i o n a l  A n u c l e o l a r transglutaminase  has been i s o l a t e d from g u i n e a  p i g l i v e r which reduces ODC a c t i v i t y by c o v a l e n t l y l i n k i n g a p u t r e s c i n e molecule t o each o f the enzyme's f o u r g l u t a m y l  s i d e chains ( 5 1 ) .  Furthermore, i t has been shown t h a t the a d d i t i o n o f the ODC-putrescine complex w i l l (52). subunit  i n c r e a s e s t o i c h i o m e t r i c a l l y , the a c t i v i t y of RNA polymerase I  There i s e v i d e n c e t o suggest t h a t t h i s complex may be the 65,000 Mr o f RNA polymerase I .  Further  e v i d e n c e o f ODC i n h i b i t i o n r e s u l t i n g  from p o s t - t r a n s l a t i o n a l m o d i f i c a t i o n has been found i n the s l i m e mold,  6.  Physarum polycephalum ( 5 3 ) . phosphorylation having ODC  by a p r o t e i n k i n a s e  activity  (54).  bovine spermatozoa and phosphorylation  I n h i b i t i o n i s achieved of Mr  through  26,000 of a p r o t e i n of Mr  S i m i l a r p r o t e i n kinases  in Ehrlich ascites cells  i s stimulated  70,000  have been found i n  (55).  by i n t e r f e r o n ( 5 6 ) .  The  latter  Others have  reported  the presence of an ODC  i n a c t i v a t i n g f a c t o r (0DIF) i n e x t r a c t s of a d u l t r a t  p r o s t a t e glands ( 5 7 ) .  The  it  mechanism of a c t i o n of ODIF i s u n c l e a r  shows a time dependent a c t i v i t y and  of h i g h l y s p e c i f i c p r o t e a s e s  spleen.  The  p o s s i b l y represent  a new  Other Lymphatic  Tissue  i s known about polyamine metabolism i n thymus  enzymes have not  been c h a r a c t e r i z e d and  i n v o l v e d i n a l t e r i n g t h e i r a c t i v i t i e s have not  ODC.  i n s i g h t i n t o the c h a r a c t e r i s t i c s and  High c o n c e n t r a t i o n s  of p u t r e s c i n e ,  found i n r a t thymus ( 5 9 ) .  been s t u d i e d  detected and  not  i n t h i s t i s s u e and the  stroma ( 6 0 ) .  which have a turnover lymphocytes elsewhere.  r e g u l a t i o n of  s p e r m i d i n e and  E x c e p t i o n a l l y h i g h ODC  in detail. offer  lymphatic  spermine have been  l e v e l s have a l s o been  are known to be a s s o c i a t e d w i t h the  The  and  the mechanisms  There i s evidence to suggest t h a t the thymus, i n p a r t i c u l a r , may valuable  class  (58).  Polyamine Metabolism i n Thymus and Relatively l i t t l e  may  although  thymus i s an a c t i v e producer of  r a t e f i v e t o t e n times h i g h e r  than t h a t  thymocytes  lymphocytes of  In the human, much of the thymic substance  undergoes a t r o p h y by the time of p u b e r t y ( 6 1 ) , w h i l e i n r a t s , i n v o l u t i o n i s known t o have taken p l a c e a t the a d u l t stage ( 6 2 ) . r e l a t i o n s h i p t h a t e x i s t s between polyamine b i o s y n t h e s i s p r o l i f e r a t i v e s t a t e of c e l l s ,  G i v e n the and  close  changes i n the  i t would seem p l a u s i b l e t h a t thymus  ODC  c o u l d undergo e x c e p t i o n a l l y l a r g e changes i n a c t i v i t y , more so than i n  7.  most other  t i s s u e s , p r i o r to a p o s i t i v e or n e g a t i v e  growth response.  The  mechanism f o r r e g u l a t i n g the enzyme's a c t i v i t y must be a b l e to accommodate the p r o d u c t i o n  of h i g h  decreases to low  l e v e l s of ODC  reported  i n r a t thymus has  i n other  not  been  While  the  studied,  lymphatic  tissues.  Studies  c a r r i e d out  on S49  lymphoma  as a m e d i a t o r of f l u c t u a t i o n s i n ODC  levels,  v a r i a n t s h a v i n g l e s i o n s i n the pathway of cAMP g e n e r a t i o n  a c t i o n w i l l not of ODC  perhaps sharp  f i n d i n g s on the c o n t r o l of t h i s enzyme's a c t i v i t y have been  c e l l s i m p l i c a t e c y c l i c AMP s i n c e S49  c e l l growth and  l e v e l s p r i o r to i n h i b i t i o n of c e l l d i v i s i o n .  mechanism of r e g u l a t i n g ODC preliminary  before  respond to any  i n w i l d type S49  cells  of the s t i m u l i t h a t produce an (63).  inhibition  Others have shown t h a t polyamine  d e p l e t i o n mediated by an i r r e v e r s i b l e and  specific  a r r e s t s the growth of mouse lymphoma c e l l s ,  but  d i s s o c i a b l e from a cAMP mediated a r r e s t ( 6 4 ) . suggest t h a t ODC  and  i n h i b i t o r of  ODC  that t h i s a r r e s t i s There i s a l s o evidence to  l e v e l s i n mouse lymphoma c e l l s may  be  regulated  i n d e p e n d e n t l y of e i t h e r p r e v i o u s l y mentioned mechanism s i n c e a  small  percentage of the c e l l s t r e a t e d w i t h t o x i c l e v e l s of the  irreversible  i n h i b i t o r of ODC  to  overproduction  The  are a b l e  of" ODC  synthesis  of a r e g u l a t o r y p r o t e i n may  demonstrate t h a t ODC concentrations  an  a l s o be r e q u i r e d  for  ODC  Experiments performed u s i n g human lymphocytes  a c t i v i t y can  be g r e a t l y reduced by the a d d i t i o n of  of i t s p r o d u c t , p u t r e s c i n e ,  spermidine, to the c u l t u r e medium ( 6 6 ) . p r o t e i n s y n t h e s i s , s i n c e the p u t r e s c i n e 47%  p r o l i f e r a t e due  (65).  c o n t r o l i n lymphocytes.  low  to s u r v i v e and  to 5% when c y c l o h e x i m i d e was  or i t s i n d i r e c t  T h i s e f f e c t appears to  product, involve  induced i n h i b i t i o n decreased from  added ( 6 7 ) .  To date, an i n h i b i t o r of  ODC  8.  has  never been r e p o r t e d  The  E f f e c t of G l u c o c o r t i c o i d s on Polyamine Metabolism i n Lymphatic As  its  a general  i n lymphatic t i s s u e .  r u l e , whenever a hormone induces an a n a b o l i c  t a r g e t t i s s u e , an e a r l y s t i m u l a t i o n of ODC  i s seen.  The  Tissue  response i n  time course  of t h i s response t y p i c a l l y shows a peak of a c t i v i t y 4 to 6 hours a f t e r the hormone i s a d m i n i s t e r e d  (68).  stimulate a generalized  net  gluconeogenesis (69).  In k i d n e y and  increase  liver,  glucocorticoids  i n protein synthesis  A p h a r m a c o l o g i c a l dose of dexamethasone has  shown to produce a r a p i d and  no e f f e c t ( 7 0 ) .  a c t i v i t y detected administration 1/2  In o t h e r  spleen  of g l u c o c o r t i c o i d s was  hours a f t e r the hormone was  thymus and  spleen  cell lysis  a f t e r hormone treatment ( 7 2 ) ,  given  reported  shown to be d e t e c t a b l e (71).  as e a r l y as  to o c c u r a p p r o x i m a t e l y 16  and  thymus weight i n r a t s (74) A negative  response r e s u l t i n g from g l u c o c o r t i c o i d treatment has demonstrated i n t i s s u e c u l t u r e s of T-lymphoblast c e l l leukemia ( 7 5 ) .  hours  and two  days  growth  a l s o been l i n e s from p a t i e n t s  To b e t t e r understand the  stages of t h i s type of growth response, two  s t u d i e s have t r i e d to  changes t h a t thymus c e l l s undergo as a r e s u l t of  with g l u c o c o r t i c o i d s .  2  Other e v i d e n c e of g l u c o c o r t i c o i d c o n t r o l of  e i g h t days r e s p e c t i v e l y , a f t e r adrenalectomy.  the e a r l i e s t  ODC  T h i s i s w e l l i n advance of  lymphocyte m i t o t i c a c t i v i t y found i n mice (73)  w i t h acute l y m p h o b l a s t i c  mineralocorticoids  of r a t s f o l l o w i n g the i n v i v o  lymphatic t i s s u e growth i s shown by the i n c r e a s e d  and  SAM  s t u d i e s , the s i g n i f i c a n t decrease i n  i n thymus and  with  been  s i g n i f i c a n t decrease i n o r n i t h i n e and  d e c a r b o x y l a s e s i n r a t thymus, whereas the i n j e c t i o n of had  associated  Two-dimensional g e l e l e c t r o p h o r e s i s has  first quantify  treatment identified  18 c o n s i s t e n t d i f f e r e n c e s when the p r o t e i n s of hormone r e s i s t a n t c e l l s  9.  were compared w i t h the g e l s o b t a i n e d a l s o reported  t h a t one  thymus c e l l s , but p o s i t i o n on  not  from s e n s i t i v e c e l l s  of the major p r o t e i n s p r e s e n t  (76).  It i s  i n the r e s i s t a n t r a t  i n the s e n s i t i v e c e l l s , m i g r a t e d to e x a c t l y the same  the 2D g e l as d i d the l a r g e s t p r o t e i n from r e s i s t a n t  lymphosarcoma c e l l s  (76).  Another s e r i e s of experiments has  the absence of a c h r o m a t o g r a p h i c a l l y thymus of dexamethasone t r e a t e d r a t s .  separable  species  When ODC  was  of ODC  partially  demonstrated i n the purified  from a crude e x t r a c t of thymus from c o n t r o l r a t s u s i n g i o n exchange chromatography, m u l t i p l e peaks .of a c t i v i t y were observed. was  This  procedure  then r e p e a t e d u s i n g a crude e x t r a c t of thymus from dexamethasone  t r e a t e d r a t s and (71).  the major peak of enzyme a c t i v i t y was  These r e s u l t s , a l t h o u g h p r e l i m i n a r y  no  longer  i n n a t u r e , suggest  present  that  g l u c o c o r t i c o i d s s i g n i f i c a n t l y a l t e r the c o n s t i t u e n t p r o t e i n s of r a t thymus and  i n one  lysis  The  i n s t a n c e , markedly a f f e c t one  of ODC  w e l l before  cell  occurs.  Present  Investigation  T h i s study was  undertaken t o determine the r e a s o n f o r the r a p i d  l a r g e d e c r e a s e i n ODC  a c t i v i t y observed i n the thymus and  dexamethasone-treated r a t s and  been p r e v i o u s l y d i s c u s s e d , decrease and  we  spleen  lytic  and  of  to i n v e s t i g a t e the r e l a t i o n s h i p of  decrease to the growth i n h i b i t i o n and  ODC  species  response of the c e l l s .  this As  has  t h e r e a r e s e v e r a l p o s s i b l e reasons f o r t h i s  chose, as a s t a r t i n g p o i n t , to l o o k f o r the presence of  an  inhibitor.  Initially, established.  the c o n d i t i o n s  These c o n d i t i o n s  for assaying  ODC  i n thymus and  s p l e e n were  i n c l u d e an e f f e c t i v e method of t i s s u e  10. p r e p a r a t i o n and c e l l d i s r u p t i o n , the optimum c o n c e n t r a t i o n s of s u l f h y d r y l r e d u c i n g agents f o r maximum ODC a c t i v i t y and s u f f i c i e n t d e t e c t the low l e v e l s o f enzyme a c t i v i t y when n e c e s s a r y .  s e n s i t i v i t y to With these  c r i t e r i a i n p l a c e , the i n f l u e n c e o f v a r i o u s f a c t o r s can be a s s e s s e d . Secondly, a r e p r o d u c i b l e assay f o r t h e presence o f an ODC i n h i b i t o r was developed.  The method chosen i s a m i x i n g a s s a y i n which ODC a c t i v i t y i s  measured s e p a r a t e l y i n a l i q u o t s o f t i s s u e e x t r a c t from c o n t r o l r a t s and from hormone-treated  r a t s , and i n m i x t u r e s o f t h e two types o f e x t r a c t s .  The presence o f an i n h i b i t o r i s i n d i c a t e d when the a c t i v i t y observed i n the m i x t u r e s i s l e s s than t h e sum o f a c t i v i t i e s i n e x t r a c t s assayed separately.  Procedures f o r i s o l a t i n g the i n h i b i t i n g  substance and  s t u d y i n g i t s c h a r a c t e r i s t i c s and p r o p e r t i e s were then developed.  Finally,  the p r o p e r t i e s o f t h i s i n h i b i t o r were compared w i t h o t h e r s d e s c r i b e d i n the  literature.  11.  MATERIALS AND  Treatment  METHODS  o f Animals  Intact  immature female W i s t a r r a t s (UBC  used i n a l l experiments.  s t r a i n ) , age 4-8 weeks, were  The animals were a l l o w e d to a d j u s t to t h e i r  surroundings f o r a t l e a s t 48 hours p r i o r t o use i n experiments.  They were  f e d w i t h s t a n d a r d l a b o r a t o r y chow and water ad l i b i t u m and kept under c o n t r o l l e d 12 hour l i g h t and 12 hour dark l i g h t i n g Dexamethasone ( H e x a d i o l phosphate) was  conditions.  d i s s o l v e d i n e t h a n o l and d i l u t e d t o  a f i n e s u s p e n s i o n i n s a l i n e immediately p r i o r t o use. a d m i n i s t e r e d a t a dose o f 200  This  was  ug by i n t r a p e r i t o n e a l i n j e c t i o n , 5 hours  p r i o r to s a c r i f i c e unless otherwise i n d i c a t e d . purposes were i n j e c t e d w i t h 0.9%  Rats used f o r c o n t r o l  s a l i n e or were not  injected.  Sample P r e p a r a t i o n The animals were k i l l e d  by c e r v i c a l d i s l o c a t i o n .  The thymus and  s p l e e n were removed immediately and p l a c e d i n i c e c o l d Hepes b u f f e r mM,  pH 7.4,  10 mM  d i t h i o t h r e i t o l and 100 MM EDTA).  (50  T i s s u e s from 3 or  more r a t s were p o o l e d and homogenized i n 2 ml o f b u f f e r per gram of t i s s u e f o r t i s s u e s from dexamethasone-treated  r a t s and 4 ml/g  from c o n t r o l r a t s .  performed u s i n g a P o t t e r - E l v e j h e m  homogenizer the 8-9  Homogenization was  tissue for tissues  w i t h the p e s t l e b e i n g d r i v e n a t a p p r o x i m a t e l y 600 rpm  up and down movements.  t h i s procedure.  The homogenizer  The r e s u l t i n g homogenate was  was  kept i n i c e throughout  c e n t r i f u g e d at 20,000 xg a t  4°C f o r 20 minutes and the s u p e r n a t a n t p o r t i o n s f i l t e r e d remove f a t and suspended  t i s s u e fragments.  dexamethasone t r e a t e d r a t s , the p e l l e t was  during  through gauze t o  F o r t i s s u e removed from then resuspended i n a volume of  12.  b u f f e r , e q u a l to the f i r s t Homogenization  was then r e p e a t e d u s i n g 4-5 up and down s t r o k e s so as to  a v o i d e x c e s s i v e foaming. filtered  e x t r a c t i o n , which c o n t a i n e d 1% L u b r o l .  The r e s u l t i n g s l u r r y was c e n t r i f u g e d and  as b e f o r e w i t h the supernatant b e i n g combined w i t h t h a t o b t a i n e d  from the f i r s t  extraction.  P e l l e t s from the e x t r a c t s of t i s s u e of c o n t r o l  r a t s were n o t e x t r a c t e d a second  ODC and I n h i b i t o r  time, except where i n d i c a t e d .  Assays  A l i q u o t s o f the s u p e r n a t a n t f r a c t i o n s were used f o r assay of ODC and SAMD.  The ODC a c t i v i t y was measured by a s l i g h t m o d i f i c a t i o n of the  p r e v i o u s method ( 7 7 ) . The r e a c t i o n m i x t u r e c o n t a i n e d , i n a f i n a l volume of  0.4 ml, 0.2 mM  of  0.136 mM  mCi/mmole).  p y r i d o x a l phosphate and L - o r n i t h i n e a t a c o n c e n t r a t i o n  c o n t a i n i n g 0.125 u C i L - l - " C o r n i t h i n e HC1 (58 1  A l l assay tubes were kept on i c e u n t i l s t a r t i n g the r e a c t i o n  w i t h the a d d i t i o n of s u b s t r a t e .  I n the a s s a y  [ '*C]C02 was 1  i n 0.2 ml hyamine h y d r o x i d e f o l l o w i n g a c i d i f i c a t i o n .  collected  Under these assay  c o n d i t i o n s , a 100 u l a l i q u o t o f thymus homogenate gave a p p r o x i m a t e l y 1200 cpm.  The method used t o d e t e c t the presence o f an ODC i n h i b i t o r took the form of a m i x i n g assay. dexamethasone-treated a 1:1 m i x t u r e .  A l i q u o t s of thymus e x t r a c t s from c o n t r o l and  r a t s were assayed s e p a r a t e l y f o r ODC and t o g e t h e r i n  The p r e d i c t e d a c t i v i t y of the m i x i n g assay would t h e r e f o r e  be the sum o f the i n d i v i d u a l ODC a c t i v i t i e s . c o u l d then be determined  The degree of i n h i b i t i o n  by comparing the observed ODC a c t i v i t y from the  m i x i n g assay w i t h the p r e d i c t e d a c t i v i t y .  One u n i t of i n h i b i t i o n was  d e f i n e d as the d i f f e r e n c e i n pmoles/100 ul/30 minutes,  between the ODC  13.  a c t i v i t y i n an a l i q u o t  of thymus ODC  from c o n t r o l r a t s and the ODC  a c t i v i t y measured i n a m i x i n g a s s a y .  The SAMD was assayed by a s l i g h t m o d i f i c a t i o n of a p u b l i s h e d method (78) c o n t a i n i n g , i n 250 ml, 0.6 mM  S-adenosyl-L-[carboxy  (60 mCi/mmole), 2.5 mM p u t r e s c i n e , 0.05 mM phosphate  11  *C]-methionine  p y r i d o x a l phosphate  and 0.1 M  b u f f e r , pH 7.1.  A l l a s s a y s were c a r r i e d out i n d u p l i c a t e o r t r i p l i c a t e and an average v a l u e used i n c a l c u l a t i o n s .  DEAE-Sepharose CL-6B Column Chromatography A s e t t l e d volume of 20.0.ml of DEAE-Sepharose CL-6B suspended i n standard Hepes b u f f e r was degassed the r e s i n was packed  f o r 2 hours.  i n a 1.5 x 10 cm. B i o r a d column t o a bed volume of  15.0 ml and washed w i t h 3 volumes of b u f f e r . i n a c o l d room a t (4°C) b e f o r e u s e . dexamethasone-treated onto the column.  A f t e r d e c a n t i n g the f i n e s  The column was  equilibrated  The thymus e x t r a c t from 6  r a t s was prepared as p r e v i o u s l y d e s c r i b e d and l o a d e d  The column was e l u t e d w i t h i n c r e a s i n g c o n c e n t r a t i o n s of  NaCl w i t h 10, 2.5 ml f r a c t i o n c o l l e c t e d a t each s t e p . of s a l t used were 0, 100 mM,  300 mM and 500 mM N a C l .  The c o n c e n t r a t i o n s Each f r a c t i o n was  assayed f o r p r o t e i n c o n t e n t u s i n g the method o f B r a d f o r d ( 7 9 ) . The f r a c t i o n s a t each s a l t c o n c e n t r a t i o n were then p o o l e d and c o n c e n t r a t e d to a p p r o x i m a t e l y 1.0 ml u s i n g u l t r a f i l t r a t i o n .  T h i s s o l u t i o n was  then  d i a l y z e d f o r 20-24 hours and then assayed f o r ODC a c t i v i t y and a b i l i t y to i n h i b i t an a l i q u o t of thymus ODC from c o n t r o l r a t s . determined by comparing  The r e c o v e r y was  the number o f i n h i b i t o r u n i t s loaded onto the  14.  column w i t h the t o t a l number of i n h i b i t o r u n i t s from each e l u t i o n . p u r i f i c a t i o n was  The  c a l c u l a t e d by comparing s p e c i f i c a c t i v i t i e s of loaded  i n h i b i t o r u n i t s w i t h t h a t o b t a i n e d from each  elution.  Heparin Sepharose Column Chromatography The H e p a r i n  Sepharose CL-6B g e l was  s w o l l e n and washed w i t h  Hepes b u f f e r f o r 15 minutes on a s i n t e r e d g l a s s f i l t e r . resuspended  i n 25 ml o f b u f f e r and  packed i n a 0.9  degassed  f o r 2 hours.  bed volumes of s t a n d a r d b u f f e r .  e q u i l i b r a t e d i n a c o l d room (4°C) b e f o r e use. to  The g e l was The  The  column  a p p l i e d t o the r e s i n .  The  column was  DEAE column w i t h i n c r e a s i n g s a l t NaCl.  The  assayed  and  With the f l o w r a t e a d j u s t e d Inhibitor  the t o t a l column volume,  was  e l u t e d i n the same manner as the  c o n c e n t r a t i o n s of 0, 100,  300  and 500  r e s u l t i n g f r a c t i o n s were p o o l e d , c o n c e n t r a t e d , d i a l y z e d  mM  and  f o r i n h i b i t o r y a c t i v i t y as b e f o r e .  S e p h a c r y l S-200 G e l  Filtration  A s e t t l e d volume of 180 ml of S e p h a c r y l S-200 was standard Hepes b u f f e r and the r e s i n was  degassed  packed i n a 1.5  volume of 100 ml.  The  f o r 2 hours.  suspended i n  A f t e r d e c a n t i n g the  x 9.0 cm Pharmacia column to a t o t a l  packed column was  washed w i t h 1 l i t r e  b u f f e r and e q u i l i b r a t e d i n a c o l d room (4°C) b e f o r e use. c a l i b r a t e d f o r m o l e c u l a r weight d e t e r m i n a t i o n purposes prepared m o l e c u l a r weight s t a n d a r d s . 10 ), F e r r i t i n 6  ml was  a minimum l e v e l , a volume of p a r t i a l l y p u r i f i e d thymus ODC  (from the DEAE Sepharose s t e p ) not exceeding  then  beads were  x 15 cm Pharmacia column t o a bed volume of 2.5  washed w i t h 10-12  standard  The  fines  bed  of standard column  was  w i t h commercialy  These i n c l u d e d Blue d e x t r a n (2 x  (440,000), C a t a l a s e (332,000), A l d o l a s e (158,000),  BSA  (67,000) and p y r i d o x a l phosphate each was  (247)..  The  sample s i z e was  1.0  ml  prepared a t a c o n c e n t r a t i o n of a p p r o x i m a t e l y 15 mg/ml.  r a t e f o r t h i s column system was  10.0  ml/hr as determined  FRAC 200 f r a c t i o n c o l l e c t o r and the p e r i s t a l t i c pump. of each 1.0  ml f r a c t i o n was  determined  The  by the  flow  Pharmacia  The p r o t e i n content  by r e c o r d i n g the  ^  e l u t i o n volume of each s t a n d a r d was  o b t a i n e d i n t h i s manner and  c a l i b r a t i o n curve was  The e l u t i o n o f each standard  constructed.  and  e  a was  c a r r i e d out u s i n g s t a n d a r d Hepes b u f f e r .  The p a r t i a l l y p u r i f i e d  thymus ODC  i n h i b i t o r p r e s e n t i n the 100  NaCl e l u t e d p o o l from the H e p a r i n Sepharose S e p h a c r y l S-200 column. was  The column was  column was  a p p l i e d t o the  e l u t e d as b e f o r e and each  assayed f o r i n h i b i t o r y a c t i v i t y u s i n g the m i x i n g assay.  The  showing h i g h i n h i b i t o r y a c t i v i t y were p o o l e d and c o n c e n t r a t e d . r e s u l t i n g volume was  mM  fraction fractions  The  then r e - a s s a y e d t o c o n f i r m i n h i b i t o r y a c t i v i t y  and  i t s e l u t i o n p o s i t i o n used t o e s t i m a t e the m o l e c u l a r weight.  M i s c e l l a n e o u s Methods a)  Trypsin Digestion;  The  t r y p s i n used i n these experiments  TPCK t r e a t e d which r e n d e r s i t f r e e of c h y m o t r y p s i n a c t i v i t y . l y o p h y l i z e d from Bovine pancreas and d i a l y z e d f r e e o f s a l t . used 1000  u n i t s of a c t i v i t y or 0.11  protein). at a 2:1 ODC mg  A n t i t r y p s i n was  mg  trypsin  was  I t has been Each  assay  (9000 BAEE u n i t s per  mg.  prepared i n the same manner as t r y p s i n and  used  r a t i o of s p e c i f i c a c t i v i t y to s t o p the a c t i o n of t r y p s i n p r i o r to  assays.  A 1.0  ml s t o c k s o l u t i o n (120 ml Hepes pH 7.6  t r y p s i n ) of t r y p s i n was  water bath.  c o n t a i n i n g 1.7  p r e p a r e d and i n c u b a t e d a t 37°C i n a shaker  At time z e r o 1.5 ml o f thymus e x t r a c t from dexamethasone  16.  t r e a t e d r a t s was added t o t h e i n c u b a t i o n m i x t u r e .  At time z e r o , 30 and 60  minutes, 500 p i a l i q u o t s o f the t r y p s i n and thymus e x t r a c t mixture was removed and i n c u b a t e d w i t h 500 u l o f a n t i t r y p s i n s t o c k s o l u t i o n (3 mg a n t i t r y p s i n / 1 . 0 ml Hepes @ pH 7.6) f o r 5 minutes.  ODC and mixing assays  were then c a r r i e d out on 100 u l a l i q u o t s o f the p o s t - a n t i t r y p s i n solution. C o n t r o l experiments were done t o show t h a t the t r y p s i n and a n t i t r y p s i n were a c t i v e .  A l i q u o t s o f enzyme e x t r a c t were combined w i t h  a l i q u o t s o f the t r y p s i n p r e p a r a t i o n and assayed f o r ODC a c t i v i t y a t v a r i o u s time i n t e r v a l s i n t h e absence  of a n t i t r y p s i n .  T h i s procedure was  repeated w i t h a n t i t r y p s i n added t o the a s s a y immediately a f t e r the a d d i t i o n of t r y p s i n .  Due t o t h e presence o f excess a n t i t r y p s i n , i t s  i n t e r a c t i o n w i t h the enzyme was a l s o a s s e s s e d .  A l i q u o t s o f the  a n t i t r y p s i n p r e p a r a t i o n were combined w i t h a l i q u o t s o f the ODC i n h i b i t o r and i n c u b a t e d f o r v a r i o u s time i n t e r v a l s . out  to v e r i f y b)  M i x i n g assays were then c a r r i e d  that ODC i n h i b i t i o n was u n a f f e c t e d .  Polyacrylamide G e l Electrophoresis.  phosphate  Stock s o l u t i o n s of sodium  b u f f e r (0.5 M, pH 7.2), SDS ( 1 0 % ) , ammonium p e r s u l f a t e  (1.5%)  and a c r y l a m i d e - b i s a c r y l a m i d e (30:0.8 p e r 100 ml) were prepared i n advance of  the sample p r e p a r a t i o n .  acrylamide.  The g e l m i x t u r e used c o n t a i n e d 10%  F o r each g e l , 42 ml o f m i x t u r e was p r e p a r e d .  14.0 ml of a c r y l a m i d e - b i s a c r y l a m i d e , 8.4 ml o f sodium  This contained  phosphate  buffer,  0.42 ml o f SDS, 2.1 ml o f ammonium p e r s u l f a t e and 17.1 ml o f d i s t i l l e d water.  T h i s m i x t u r e was then degassed  TEMED was added. phosphate  f o r 10 min a t which  time 21 u l o f  The c o n t i n u o u s b u f f e r system used 200 ml sodium  b u f f e r (0.5 M sodium  and 790 ml o f d i s t i l l e d water  phosphate  pH 7.2 per l i t r e ) 10 ml SDS (10%)  ( p e r l i t r e ) as an e l e c t r o d e  buffer.  17.  For a 50-100 ug p r o t e i n sample, a s o l u t i o n o f 10 u l sodium phosphate  b u f f e r , 100 PI SDS, 25 Ml 6-mercaptoethanol  ( 5 % ) , 50 y l  g l y c e r o l (10%) and 50 u l bromophenol b l u e (0.2%) was b o i l e d f o r 3 minutes.  T h i s sample was then c e n t r i f u g e d a t 20,000 xg f o r 5 minutes.  Samples of 5 u l and 10 u l were loaded onto a l t e r n a t e l a n e s o f the gel. was  E l e c t r o p h o r e s i s was c a r r i e d out a t 140 v o l t s f o r 6 hours.  The g e l  s t a i n e d o v e r n i g h t i n a 5:5:2 m i x t u r e o f Coomassie blue ( 0 . 1 % ) ,  methanol and g l a c i a l a c e t i c a c i d . 20% methanol and 7% a c e t i c  c)  Ultrafiltration.  The d e s t a i n i n g was c a r r i e d out u s i n g  acid.  T h i s procedure was c a r r i e d out u s i n g t h e Amicon  D i a f l o YM10, YM30 o r XM50 membranes.  F o r m o l e c u l a r weight  minimum e x t r a c t volume o f 10.0 ml was used. c a r r i e r gas a t a p r e s s u r e o f 80 p s i .  estimations, a  N i t r o g e n was used as a  The f i n a l r e t e n t a t e volume was  a p p r o x i m a t e l y 1.0 ml. and t h e f i l t r a t e was c o l l e c t e d i n a graduated c y l i n d e r t o monitor t h e volume r e d u c t i o n . fractions,  F o r c o n c e n t r a t i o n o f column  t h e YM10 membrane was used as d e s c r i b e d above. A l l  u l t r a f i l t r a t i o n procedures were c a r r i e d out a t 4°C.  d)  Dialysis.  A l l d i a l y s i s t u b i n g was p r e p a r e d i n advance by b o i l i n g  f o r 1 hour i n a s o l u t i o n o f d i l u t e a c e t i c a c i d and EDTA.  I t was c u t t o  s i z e and washed t h o r o u g h l y w i t h d i s t i l l e d water and then b u f f e r immediately 7.4).  p r i o r to use.  The d i a l y s i s b u f f e r used was Hepes (50 mM pH  The d i a l y s i s b u f f e r volume used was a t l e a s t 500 x g r e a t e r than the  sample volume and a minimum o f t h r e e b u f f e r changes were used over a 20-24 period.  S t i r r i n g was c o n s t a n t l y m a i n t a i n e d and a l l d i a l y s i s took p l a c e a t  18.  8.  Chemicals D i t h i o t r e i t o l , p y r i d o x a l phosphate, L - o r n i t h i n e , p u t r e s c i n e and  t r y p s i n (TPCK-treated) were purchased from Sigma Chemical Company, S t . L o u i s , MO, USA.  S-adenosylmethionine was o b t a i n e d  Calbiochem-Behring, San Diego, CA, USA. from Amersham C o r p o r a t i o n ,  I L , USA.  from  L - t l - " C ] O r n i t h i n e was 1  obtained  19.  RESULTS  Conditions  f o r Measurement o f ODC  Experiments were f i r s t  Activity  d i r e c t e d toward e s t a b l i s h i n g assay  conditions  s u i t a b l e f o r measuring ODC a c t i v i t y i n t i s s u e s o f c o n t r o l animals. primary importance was o p t i m i z i n g measurable r a d i o a c t i v i t y . ODC a c t i v i t i e s o b t a i n e d assaying as  CO2 p r o d u c t i o n  and  therefore  The d a t a p r e s e n t e d i n T a b l e 1 demonstrates the  from homogenizing thymus from c o n t r o l r a t s and  enzyme a c t i v i t y w i t h d i f f e r e n t b u f f e r systems.  the s t a n d a r d  activity.  Hepes was chosen  b u f f e r because i t c o n s i s t e n t l y gave the g r e a t e s t  ODC i n most t i s s u e s r e q u i r e s reduced s u l f h y d r y l groups f o r  maximum a c t i v i t y . concentrations  The r e s u l t s i l l u s t r a t e d  i n T a b l e 2 show t h a t 10 mM  of d i t h i o t h r e i t o l were optimum f o r the amount of thymus  enzyme, a l t h o u g h lower c o n c e n t r a t i o n s activity.  Of  still  a l l o w e d measurable enzyme  I t i s a l s o n e c e s s a r y t o determine a s u b s t r a t e  t h a t w i l l a v o i d problems a s s o c i a t e d w i t h s u b s t r a t e  concentration  limitation.  L - o r n i t h i n e k i n e t i c s of crude thymus ODC a r e i l l u s t r a t e d  The  i n F i g u r e 1.  The  double r e c i p r o c a l p l o t gave an apparent Km f o r o r n i t h i n e of 80 pM which i s w i t h i n the range r e p o r t e d  f o r ODC from o t h e r  the a s s a y c o n d i t i o n s a r e d e s c r i b e d Figures  assay c o n d i t i o n s .  The d e t a i l s of  i n the methods s e c t i o n .  2 and 3 show t h a t thymus and s p l e e n  i s l i n e a r with respect  sources.  The r e s u l t s I n  ODC a c t i v i t y i n crude e x t r a c t s  t o time and p r o t e i n c o n c e n t r a t i o n  under optimum  T h i s was a l s o shown t o be t r u e f o r ODC a c t i v i t y i n  e x t r a c t s of t i s s u e s taken from dexamethasone t r e a t e d r a t s .  TABLE 1 ODC ACTIVITY IN THYMUS EXTRACTS PREPARED USING DIFFERENT BUFFERS The r a t s used were e i t h e r s a l i n e i n j e c t e d o r n o n - i n j e c t e d . Each b u f f e r was prepared a t a c o n c e n t r a t i o n o f 50 mM and a d j u s t e d t o pH 7.4. ODC assays were then conducted as d e s c r i b e d i n the Methods s e c t i o n . A l l assays were c a r r i e d out i n d u p l i c a t e o r t r i p l i c a t e and t h e r e s u l t s averaged.  BUFFER* (mM)  *  see l i s t  ODC ACTIVITY (pmoles ^COp/lOO Ml/30 min.)  HEPES  206  HEPPS  185  MOPS  189  o f a b b r e v i a t i o n s f o r IUPAC nomenclature o f b u f f e r s  21. TABLE 2 ODC ACTIVITY IN THYMUS EXTRACTS OF CONTROL RATS DETERMINED USING DIFFERENT DITHIOTHREITOL CONCENTRATIONS ODC assays were c a r r i e d out as d e s c r i b e d i n the Methods s e c t i o n . d i t h i o t h r e i t o l c o n c e n t r a t i o n was a d j u s t e d t o the d e s i r e d l e v e l i n the r e a c t i o n volume of the a s s a y .  DITHIOTHREITOL CONCENTRATION (mM)  ODC ACTIVITY (pmoles/100 jil/30 min)  3  285  5  269  10  471  15  317  The  FIGURE 1 L-ORNITHINE KINETICS OF ODC IN THYMUS OF CONTROL RATS ODC a c t i v i t y i n thymus e x t r a c t s o f c o n t r o l r a t s was determined a t various substrate concentrations. A l l a s s a y s were c a r r i e d out I n t r i p l i c a t e and t h e r e s u l t s averaged. Km = 80 MM Vmax = 1.6 x 1 0  s  pmoles ' " ^ / l O O Ml/30 min.  23. FIGURE 2 EFFECT OF INCUBATION  TIME ON THYMUS ODC ACTIVITY  A l i q u o t s of thymus e x t r a c t from (o) c o n t r o l r a t s and from ( o ) dexamethasone-treated r a t s were assayed f o r ODC a c t i v i t y . The assays were stopped a t the times i n d i c a t e d . A l l a s s a y s were c a r r i e d out i n d u p l i c a t e or t r i p l i c a t e .  23a.  24. FIGURE 3 EFFECT OF PROTEIN CONCENTRATION ON THYMUS ODC  ACTIVITY  The ODC a c t i v i t y was measured i n thymus e x t r a c t s of (o) c o n t r o l r a t s and of ( o ) dexamethasone-treated r a t s u s i n g d i f f e r e n t volumes of each extract. The d e t a i l s of the a s s a y procedure a r e d e s c r i b e d i n the methods section.  25.  The Occurrence of an I n h i b i t o r of ODC i n Lymphatic  T i s s u e from  Dexamethasone T r e a t e d R a t s . The p o s s i b i l i t y t h a t the r a p i d decrease i n ODC a c t i v i t y i n thymus and s p l e e n i n dexamethasone t r e a t e d  rats resulted  i n h i b i t o r o r i n a c t i v a t o r was f i r s t  from the p r o d u c t i o n of an  i n v e s t i g a t e d by measuring  the ODC  a c t i v i t y o b t a i n e d from m i x i n g a l i q u o t s of t i s s u e e x t r a c t from c o n t r o l and dexamethasone-treated  rats.  The r e s u l t s i n T a b l e 3a show that the  observed enzyme a c t i v i t y was c o n s i d e r a b l y l e s s than the sum of a c t i v i t i e s i n e x t r a c t s assayed i n d i v i d u a l l y .  In a s e r i e s of t e n s i m i l a r  the observed a c t i v i t y ranged from 28% to 61% of the p r e d i c t e d v a l u e i n thymus and 22% t o 54% i n s p l e e n .  additive  S i n c e , i n most c a s e s , the  a c t i v i t y c o n t r i b u t e d by the e x t r a c t from dexamethasone-treated l e s s than 10% of the t o t a l ,  experiments,  animals was  d e s t r u c t i o n o r i n h i b i t i o n of ODC i n e x t r a c t s  from c o n t r o l animals must have o c c u r r e d .  Prompted by the wide range o f  i n h i b i t i o n o r i n a c t i v a t i o n o f ODC observed i n these mixing a s s a y s , the p e l l e t s r e s u l t i n g from the two t i s s u e p r e p a r a t i o n s were assayed f o r ODC a c t i v i t y and a b i l i t y t o i n h i b i t an a l i q u o t of enzyme p r e p a r a t i o n from c o n t r o l animals.  The d a t a p r e s e n t e d i n T a b l e 2b show t h a t l e s s than 5% o f  the t o t a l ODC a c t i v i t y remained  i n the p e l l e t s o f s p l e e n and thymus  e x t r a c t s from n o n - i n j e c t e d o r c o n t r o l a n i m a l s .  However, i t was found  a s u b s t a n t i a l amount o f the i n h i b i t o r y substance remained resuspended  i n the  p e l l e t s of thymus and s p l e e n from dexamethasone-treated  Experiments were then d e s i g n e d t o s t u d y the r e l e a s e of i n h i b i t o r pelleted material. still  left  that  rats.  from  A second e x t r a c t i o n of the p e l l e t s w i t h Hepes b u f f e r ,  inhibitory activity unsolubilized.  try e x t r a c t i o n with detergent.  I t was t h e r e f o r e d e c i d e d to  L u b r o l i s known t o be an e f f e c t i v e  f o r s o l u b i l i z i n g a v a r i e t y o f p r o t e i n s ( 8 1 ) . I n i t a l l y each p e l l e t  agent was  26.  TABLE 3 INHIBITION OF ODC ACTIVITY BY AQUEOUS EXTRACTS OF THYMUS AND SPLEEN FROM DEXAMETHASONE-TREATED RATS Rats r e c e i v e d e i t h e r s a l i n e ( c o n t r o l group) o r dexamethasone (200 Mg) 5 hours b e f o r e s a c r i f i c e . T i s s u e s were pooled from 3-4 r a t s per group. Enzyme a c t i v i t y was assayed i n d u p l i c a t e o r t r i p l i c a t e u s i n g 100 Ml a l i q u o t s o f each t i s s u e p r e p a r a t i o n . T i s s u e e x t r a c t s were prepared as d e s c r i b e d i n t h e methods s e c t i o n .  a)  BUFFER EXTRACTION OF THYMUS AND SPLEEN ODC A c t i v i t y (pmoles ''COp/lOO Ml/30 min.)  Source o f T i s s u e i n Assay Treatment of Animals 1. 2. 3. 4. 5. 6. 7. 8.  b)  Control Control Hormone T r e a t e d Hormone T r e a t e d  Observed  Predicted  Thymus Spleen Thymus Spleen 1 + 3 2 + 4 1 + 4 2 + 3  237 140 13 15 127 70 140 91  -  -  250 155 252 153  51 46 56 60  BUFFER EXTRACTION OF PELLETED MATERIAL ODC A c t i v i t y (pmoles " C 0 / 1 0 0 ul/30 min.)  Source o f T i s s u e i n Assay Treatment o f Animals 1. 2. 3.  Control Control Hormone T r e a t e d  4.  Hormone T r e a t e d  5.  Control  6.  Control  7. 8.  % Predicted  Tissue  ,  2  % Predicted  Tissue  Observed  Predicted  Thymus Spleen Thymus Pellet Spleen Pellet Thymus Pellet Spleen Pellet  252 189 13  —  —  -  -  12  -  -  4  -  -  4  -  -  1 + 3 2 + 4  185 200  265 264  70 76  e x t r a c t e d w i t h a volume of Hepes b u f f e r , e q u a l to the f i r s t c o n t a i n i n g 1% L u b r o l .  extraction,  These two e x t r a c t i o n volumes were then combined and  assayed f o r i n h i b i t o r y a c t i v i t y .  The d a t a l i s t e d  i n Table 4  the r e s u l t s of m i x i n g experiments  c a r r i e d out u s i n g a l i q u o t s of L u b r o l  e x t r a c t s and of b u f f e r e x t r a c t s from c o n t r o l a n i m a l s . percentage o f p r e d i c t e d a c t i v i t y r e c o v e r e d ranged 81-86% i n h i b i t i o n of ODC. 66-73% ODC i n h i b i t i o n .  demonstrate  In the s p l e e n , the  from 14-19%, s i g n i f y i n g  In the thymus, the range was 27-34% s i g n i f y i n g  To determine whether the i n c r e a s e d i n h i b i t i o n  observed a f t e r L u b r o l e x t r a c t i o n was due t o the volume of a d d i t i o n a l i n h i b i t o r or to d i r e c t  i n h i b i t i o n of ODC by L u b r o l i t s e l f ,  a s e r i e s of  experiments were c a r r i e d out w i t h L u b r o l , and thymus and s p l e e n from c o n t r o l animals.  Initially,  thymus and s p l e e n e x t r a c t s were prepared  c o n t r o l animals u s i n g Hepes b u f f e r o n l y and assayed  f o r ODC  Then assays were c a r r i e d out on t h e s e e x t r a c t s a f t e r c o n c e n t r a t i o n s of L u b r o l were added.  from  activity.  different  The r e s u l t s i l l u s t r a t e d  i n Figure 4  show t h a t L u b r o l added d i r e c t l y t o the a s s a y , a t c o n c e n t r a t i o n s of up to 2%, has l i t t l e o r no i n h i b i t o r y e f f e c t on thymus ODC.  However, an  i n h i b i t o r y o r i n a c t i v a t i n g e f f e c t o f a p p r o x i m a t e l y 30% i s observed i n s p l e e n ODC assays a t L u b r o l c o n c e n t r a t i o n s of g r e a t e r than 0.1%. s e r i e s of experiments  A second  was c a r r i e d out i n c o r p o r a t i n g d i f f e r e n t L u b r o l  c o n c e n t r a t i o n s i n t o the b u f f e r p r i o r t o t i s s u e e x t r a c t i o n .  ODC  assays  were then c a r r i e d out w i t h t h e r e s u l t s b e i n g the same as i n the f i r s t L u b r o l experiments demonstrate  ( d a t a not shown).  some s o r t o f ODC i n h i b i t i o n o r i n a c t i v a t i o n w e l l i n excess of  30% i n s p l e e n as a r e s u l t removal  T h e r e f o r e , w h i l e i t was p o s s i b l e t o  of dexamethasone treatment, the e f f e c t i v e  o f L u b r o l ( t o c o n c e n t r a t i o n s of l e s s than 0.1%) a f t e r  tissue  e x t r a c t i o n w i t h d e t e r g e n t i s n e c e s s a r y f o r f u r t h e r i n v e s t i g a t i o n of the  28.  TABLE 4 OCCURRENCE OF AN INHIBITOR OF THYMUS AND SPLEEN ODC IN LUBROL EXTRACTED LYMPHOID TISSUE FROM DEXAMTHASONE-TREATED RATS Rats r e c e i v e d e i t h e r s a l i n e ( c o n t r o l group) o r dexamethasone (200 Mg) 5 hours b e f o r e s a c r i f i c e . T i s s u e s were pooled from 3-4 r a t s p e r group. Enzyme a c t i v i t y was assayed i n d u p l i c a t e o r t r i p l i c a t e u s i n g 100 Ml a l i q u o t s o f each t i s s u e p r e p a r a t i o n . T i s s u e e x t r a c t s were prepared as d e s c r i b e d i n t h e Methods s e c t i o n w i t h the second e x t r a c t i o n b u f f e r c o n t a i n i n g 1% L u b r o l .  Source o f T i s s u e  i n Assay  Treatment o f Animals  ODC A c t i v i t y Tissue  Observed  (pmoles  1  "CO^/lOO M.l/30 min.)  Predicted  %  Predicted  1.  Control  Thymus  235  -  -  2.  Control  Spleen  122  -  -  3.  Hormone  Treated  Thymus  12  -  -  4.  Hormone  Treated  Spleen  11  -  -  5.  -  1 + 3  82  247  30  6.  -  2 + 4  24  133  18  7.  -  1 + 4  86  246  35  FIGURE A THE EFFECT OF LUBROL ON ODC ACTIVITY IN RAT THYMUS AND  SPLEEN  t  L u b r o l was i n c o r p o r a t e d i n t o the assay m i x t u r e e i t h e r d i r e c t l y or a t the time of t i s s u e homogenization. The d e t a i l s o f t h i s procedure a r e d i s c u s s e d i n the Methods s e c t i o n . A l l a s s a y s were performed i n d u p l i c a t e or t r i p l i c a t e , a) ( o ) ODC a c t i v i t y i n thymus o f c o n t r o l r a t s , b) (o) ODC a c t i v i t y i n s p l e e n of c o n t r o l r a t s .  8  800  1.5  2.0  0.5  Lubrol Concentration (%)  1.0  30.  s p l e e n system. toward  F o r t h i s r e a s o n , the m a j o r i t y o f experiments  directed  c h a r a c t e r i z i n g t h e i n h i b i t o r y substance were c a r r i e d out u s i n g  thymus e x t r a c t s from c o n t r o l animals as a source o f enzyme.  I n h i b i t i o n was shown t o be independent spleen.  o f time i n both thymus and  The e x p e r i m e n t a l r e s u l t s d e s c r i b e d i n T a b l e 5 show t h a t a t the  i n c u b a t i o n times i n v e s t i g a t e d , maximum i n h i b i t i o n was reached a t t h e earliest  time i n t e r v a l i r r e s p e c t i v e o f t h e r e l a t i v e p r o p o r t i o n s o f ODC  from c o n t r o l t i s s u e s and e x t r a c t s p r e p a r e d from rats.  dexamethasone-treated  When mixing a s s a y s u s i n g thymus e x t r a c t s were c a r r i e d out a t  different remained  s u b s t r a t e c o n c e n t r a t i o n s , t h e degree o f i n h i b i t i o n observed constant.  The r e s u l t s l i s t e d  i n T a b l e 6 show that 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 by a f a c t o r o f 50 d i d n o t change the i n h i b i t i o n observed.  The a f f i n i t y f o r o r n i t h i n e demonstrated  by the a c t i v e  enzyme  molecules i n t h e m i x i n g a s s a y was n e a r l y i d e n t i c a l t o t h a t demonstrated by ODC m o l e c u l e s i n thymus e x t r a c t s from c o n t r o l r a t s .  The double  reciprocal  p l o t i n F i g u r e 5 a l s o shows t h a t t h e Vmax f o r t h e 1:1 mixtures o f t i s s u e a l i q u o t s i s a p p r o x i m a t e l y one e i g h t h o f t h a t measured f o r enzyme from control  rats.  As shown i n T a b l e 7, t h e i n h i b i t o r y substance was d e t e c t a b l e i n thymus as e a r l y as 2.5 hours a f t e r treatment, but c o u l d not be d e t e c t e d i n r a t s t r e a t e d 24 hours p r i o r t o s a c r i f i c e . substance appeared  The l e v e l s o f i n h i b i t o r y  t o be h i g h e s t a t 5 and 12 hours a f t e r hormone  injection.  The degrees, o f thymus ODC i n h i b i t i o n was shown t o v a r y i n d i r e c t p r o p o r t i o n t o t h e volume o f thymus e x t r a c t from dexamethasone-treated  rats  31.  TABLE 5 INHIBITION OF ODC IN THYMUS AND SPLEEN OF DEXAMETHASONE-TREATED RATS AS A FUNCTION OF INCUBATION  TIME  E x t r a c t s from thymus and s p l e e n were prepared as d e s c r i b e d i n the Methods s e c t i o n . The p r o p o r t i o n s of enzyme and i n h i b i t o r e x t r a c t s were v a r i e d i n the r a t i o s d e s c r i b e d . A l l assays were done i n t r i p l i c a t e and the r e s u l t s averaged.  a)  RATIO OF ENZYME TO INHIBITOR OF 1:1  Time of Incubation (minutes)  15 30 45 60  b)  % Inhibition of Thymus ODC  70 66 73 70  % Inhibition of Spleen ODC  80 81 86 83  RATIO OF ENZYME TO INHIBITOR OF 2.5:1  Time o f Incubation (minutes)  15 30 45 60  % Inhibition of Thymus ODC  27 28 29 32  % Inhibition of Spleen ODC  56 52 57 56  TABLE 6 ACTIVITY OF THYMUS ODC  INHIBITOR AT DIFFERENT SUBSTRATE CONCENTRATIONS  E x t r a c t s of thymus from s a l i n e ( c o n t r o l ) and hormone-treated (200 ug dexamethasone) a n i m a l s were prepared 5 hours a f t e r i n j e c t i o n as d e s c r i b e d i n the Methods s e c t i o n . T i s s u e s were pooled from 3-5 animals per group. ODC i n h i b i t o r y a c t i v i t y was measured as d e s c r i b e d i n the Methods s e c t i o n a t the d i f f e r e n t o r n i t h i n e c o n c e n t r a t i o n s l i s t e d , u s i n g d u p l i c a t e o r t r i p l i c a t e measurements f o r each sample.  Ornithine concentration i n assay (mM) 10  ODC A c t i v i t y , pmoles Co2/30 min i n e x t r a c t of thymus from Controls 101  Hormone-Treated 2  Mixture 32  % Inhibition 31  50  236  7  74  27  100  292  8  86  29  250  373  13.  127  32  500  426  21  136  31  33.  FIGUEE 5 L-ORNITHINE KINETICS OF THYMUS ODC IN A MIXING ASSAY (o) The ODC a c t i v i t y i n a 1:1 m i x t u r e o f thymus e x t r a c t from c o n t r o l r a t s and from dexamethasone-treated r a t s was determined a t v a r i o u s s u b s t r a t e c o n c e n t r a t i o n s and shown t o have a near i d e n t i c a l Km f o r o r n i t h i n e t o ODC i n thymus e x t r a c t s from c o n t r o l r a t s ( o ) . Km = 80 uM Vmax = 1.6 x 10  pmoles "C0 /100 pl/30 min  3  Vmaxj = 0.2 x 1 0  l  2  3  pmoles "C0 /100 nl/30 min 1  2  TABLE 7 ACTIVITY OF ODC AND ODC INHIBITOR IN THYMUS EXTRACTS PREPARED FROM RATS AT DIFFERENT TIMES AFTER DEXAMETHASONE TREATMENT Thymus e x t r a c t s were p r e p a r e d from r a t s (3-4 per group) at times i n d i c a t e d a f t e r treatment w i t h dexamethasone. ODC a c t i v i t y was measured i n d u p l i c a t e i n 100 u l of each e x t r a c t and ODC i n h i b i t o r y a c t i v i t y was determined i n a m i x i n g a s s a y as d e s c r i b e d i n the Methods s e c t i o n .  Time A f t e r  Treatment  (hours)  Specific Activity of ODC % of C o n t r o l  % I n h i b i t i o n of ODC in M i x i n g Assay  2.5  32  12.3  5.0  4  71.0  12.0  5  48.0  24.0  15  0  added t o the m i x i n g assay.  The r e s u l t s d e s c r i b e d by T a b l e 8 a l s o show  t h a t when t h e volume of i n h i b i t o r - c o n t a i n i n g the volume o f enzyme-containing e x t r a c t  extract  varied,  was kept c o n s t a n t and  a near l i n e a r decrease i n  i n h i b i t i o n was observed.  Characterization  o f ODC I n h i b i t i n g Substance i n Thymus of  Dexamethasone-Treated  Rats.  E x p e r i m e n t a l r e s u l t s suggest t h a t thymus e x t r a c 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 crude  from dexamethasone-treated  s p e c i f i c f o r ODC.  rats  i s at least .partially  Assays o f SAMD i n crude thymus e x t r a c t s  o f c o n t r o l and  dexamethasone-treated r a t s showed 86% o f p r e d i c t i v e a d d i t i v e v a l u e i n a mixing experiment. the  E x p e r i m e n t a l r e s u l t s show how t h i s r e s u l t d i f f e r s  from  r e c o v e r y o f ODC u s i n g t h e same t i s s u e p r e p a r a t i o n s (data not shown).  In a m i x i n g experiment, o n l y 39% o f t h e p r e d i c t e d observed.  When combined w i t h o t h e r s o u r c e s o f ODC, t h e i n h i b i t i n g  substance c o n t a i n e d i n thymus e x t r a c t s displayed  ODC a c t i v i t y was  from dexamethasone-treated  a s p e c i f i c i t y f o r enzyme from l y m p h a t i c t i s s u e .  thymus e x t r a c t  from dexamethasone t r e a t e d  An a l i q u o t o f  r a t s w i l l i n h i b i t thymus ODC  from c o n t r o l r a t s by 70%, w h i l e i t w i l l i n h i b i t spleen extract  rats  ODC i n an a l i q u o t o f  from c o n t r o l r a t s by 60% and ODC from r a t k i d n e y by o n l y  30% ( d a t a n o t shown).  When t h e thymus e x t r a c t  from dexamethasone-treated animals was heated  a t 55°C f o r 2 minutes, a l l o w e d t o c o o l and then mixed w i t h an a l i q u o t o f thymus e x t r a c t  from c o n t r o l a n i m a l s , t h e r e was no i n h i b i t i o n observed  ( d a t a not shown).  The crude thymus e x t r a c t s  from  dexamethasone-treated  r a t s were i n c u b a t e d w i t h v a r i o u s enzymes which degrade p r o t e i n s  and/or  36.  TABLE 8 EFFECTS OF VARYING THE RATIO OF INHIBITOR TO ENZYME ON ODC ACTIVITY E x t r a c t s o f thymus were p r e p a r e d from c o n t r o l r a t s (as source o f enzyme) and from dexamethasone-treated r a t s ( s o u r c e of i n h i b i t o r ) . T i s s u e s were pooled from 4-6 a n i m a l s per group. ODC i n h i b i t i o n was measured as d e s c r i b e d i n the t e x t u s i n g d i f f e r e n t amounts of the two extracts. Assays were done i n d u p l i c a t e o r t r i p l i c a t e .  Amount  of Enzyme  ul  Amount  of I n h i b i t o r  % Inhibition of ODC  ul  100  0  .  0  100  50  37  100  100  56  100  250  72  50  100  100  100  56  250  100  34  "  73  37.  n u c l e i c a c i d s and then re-examined activity. appeared  for their ability  E x p e r i m e n t a l r e s u l t s show t h a t the i n h i b i t o r y to be u n a f f e c t e d by n u c l e a s e s .  DNase f o r 60 minutes d i d not r e s u l t inhibit  to i n h i b i t  thymus ODC  for a b i l i t y  I n c u b a t i o n w i t h e i t h e r RNAse or  from c o n t r o l r a t s ( d a t a not shown).  The s e n s i t i v i t y o f  a s s e s s e d a t v a r i o u s times a f t e r  The e x t r a c t c o n t a i n i n g the i n h i b i t i n g substance was to i n h i b i t  thymus ODC  incubation with t r y p s i n . i n h i b i t ODC  substance  i n a s i g n i f i c a n t change i n a b i l i t y to  the i n h i b i t o r y substance t o t r y p s i n was incubation.  from c o n t r o l r a t s b e f o r e and  after  The r e s u l t s i n F i g u r e 6 show t h a t the a b i l i t y t o  substance has been shown to be n o n - d i a l y z a b l e and not  dependent on a s m a l l m o l e c u l a r weight  substance f o r a c t i v i t y .  T a b l e 9 show t h a t when thymus e x t r a c t s from both c o n t r o l r a t s dexamethasone-treated  The data i n and  r a t s were d i a l y z e d s e p a r a t e l y b e f o r e a mixing  the degree o f i n h i b i t i o n d e t e c t e d i n a m i x i n g assay was  same as t h a t observed u s i n g u n d i a l y z e d e x t r a c t s . u l t r a f i l t r a t i o n were conducted i n h i b i t o r y substance.  to approximate  r e s u l t s of m i x i n g experiments i n T a b l e 9.  P u r i f i c a t i o n of ODC  Experiments u s i n g  the m o l e c u l a r weight of the  the i n h i b i t i n g substance.  The  conducted on the f i l t r a t e and r e t e n t a t e are  In a l l c a s e s , o n l y the r e t e n t a t e demonstrated  a b i l i t y to i n h i b i t ODC  the  Membranes w i t h s o l u t e r e t e n t i o n l e v e l s of 10,000,  30,000 and 50,000 d a l t o n s , a l l r e t a i n e d  listed  checked  d e c r e a s e d markedly w i t h c o n t i n u i n g exposure t o t r y p s i n .  The i n h i b i t i n g  experiment,  ODC  from the thymus o f c o n t r o l  an  rats.  Inhibitor  The i n i t i a l attempts t o p u r i f y the ODC chromatography on i o n exchange columns.  inhibitor  involved  These i n c l u d e d DEAE-Sepharose and  38. FIGURE 6 EFFECT OF TRYPSIN ON ACTIVITY OF THYMUS ODC  INHIBITOR  I n h i b i t i o n of ODC was determined by m i x i n g a s s a y s . A l l assays were done i n t r i p l i c a t e and t h e r e s u l t s averaged. The d e t a i l s o f the t r y p s i n procedure a r e d e s c r i b e d i n the Methods s e c t i o n .  33a.  TABLE 9 EFFECT.OF SMALL MOLECULAR WEIGHT SUBSTANCES ON THYMUS ODC INHIBITORY ACTIVITY The d i a l y s i s and u l t r a f i l t r a t i o n procedures used a r e d e s c r i b e d i n t h e Methods s e c t i o n . A l l a s s a y s were c a r r i e d out i n t r i p l i c a t e and r e s u l t s averaged.  a)  1. 2. 3. 4. 5. 6.  b)  DIALYSIS Treatment of E x t r a c t  Source o f Tissue Extract  Pre D i a l y s i s Pre D i a l y s i s  Control Hormone-treated 1 + 2 Control Hormone-treated 4 + 5  Post D i a l y s i s Post D i a l y s i s  ODC A c t i v i t y % Inhibition '"COp/lOO pl/30 min) o f Thymus ODC 173 7 66 174 10 74  37  40  ULTRAFILTRATION  Treatment of E x t r a c t  Source of Tissue Extract  1. 2. YM-30 R e t e n t a t e 3. YM-30 F i l t r a t e 4. 5. 6. XM-50 R e t e n t a t e 7. XM-50 F i l t r a t e 8. 9.  (pmoles  Control Hormonetreated Hormonetreated 1 + 2 1 + 3 Hormonetreated Hormonetreated 1 + 6 1 + 7  ODC A c t i v i t y (pmoles "CO^/lOO U.l/30 min) 1  % Inhibition of Thymus ODC A c t i v i t y  152 9  89 157 12  44 Nil  49 166  70 Nil  40.  phosphocellulose. substance  P r e l i m i n a r y experiments i n d i c a t e d that the  contained  i n detergent  inhibiting  e x t r a c t s of the thymus homogenate from  dexamethasone-treated r a t s bound o n l y m a r g i n a l l y to p h o s p h o c e l l u l o s e , nearly completely first  to DEAE Sepharose CL-6B.  attempted by step-wise  found  that thymus ODC  was  c o n c e n t r a t i o n s of 50 mM, 150 mM  E l u t i o n of the DEAE column  i n c r e a s e s of i o n i c s t r e n g t h .  the c o n t r o l experiments p r i o r to b e g i n n i n g inhibited thymus ODC  F o r t h i s reason,  a c t i v i t y i s i n h i b i t e d by 20% and 56%.  immediately  e l u t i o n , the f r a c t i o n s were p o o l e d approximately  1.0  ml.  i t was  F i g u r e 7 shows t h a t at NaCl  and  T h i s volume was  activity  a f t e r each step of NaCl  concentrated  to a f i n a l volume of  then d i a l y z e d o v e r n i g h t and  assays were c a r r i e d out the f o l l o w i n g day  t o determine  mixing  inhibitory  activity.  The  s a l t c o n c e n t r a t i o n s used t o e l u t e the p r o t e i n were 0,  mM,  and  500 mM  300 mM  NaCl.  at  T h i s f i n d i n g made the  a s s a y i n g of i n d i v i d u a l column f r a c t i o n s f o r i n h i b i t o r y impractical.  P r o t e i n a s s a y s were c a r r i e d out on  f r a c t i o n s throughout the e n t i r e s a l t g r a d i e n t .  100  individual  The f u n c t i o n a l  d i s t r i b u t i o n of p r o t e i n e l u t e d from the DEAE column i s shown i n Table  The  l a r g e m a j o r i t y of ODC  NaCl e l u t i o n volume.  The  i n h i b i t o r y a c t i v i t y found e l u t i o n step. estimate  i n h i b i t o r y a c t i v i t y was  The  i n the v a r i o u s c o n c e n t r a t e d  Polyacrylamide  300 mM  g e l e l e c t r o p h o r e s i s was  i n the 300  volumes from each then used to  i n each p o o l of e l u t e d  NaCl p o o l showed s i x bands.  were a s i n g l e band t h a t m i g r a t e d d a l t o n s and  found  10.  mM  r e s u l t s i n T a b l e 10 o u t l i n e the d i s t r i b u t i o n of  the number of p r o t e i n s c o n t a i n e d  fractions.  was  In c a r r y i n g out  the DEAE procedure,  by s a l t .  NaCl, i n h i b i t i o n i s a p p r o x i m a t e l y  but  The  darkest  to a p o s i t i o n c o r r e s p o n d i n g  a p a i r of bands a t a p p r o x i m a t e l y  60,000 d a l t o n s .  staining  to 120,000 There  was  FIGURE 7 EFFECT OF SODIUM CHLORIDE ON THYMUS ODC ACTIVITY ODC a c t i v i t y was a s s e s s e d a t v a r i o u s s a l t c o n c e n t r a t i o n s . These a c t i v i t i e s were then compared w i t h t h e ODC a c t i v i t y measured i n the absence o f s a l t . A l l assays were c a r r i e d out i n d u p l i c a t e o r t r i p l i c a t e .  Percent Inhibition of O D C Activity  TABLE 10 PURIFICATION OF THYMUS ODC INHIBITOR USING DEAE SEPHAROSE CL-6B COLUMN CHROMATOGRAPHY The column c o n d i t i o n s used i n t h i s procedure a r e d i s c u s s e d i n t h e Methods s e c t i o n . The f r a c t i o n s e l u t e d a t each NaCl c o n c e n t r a t i o n were pooled and c o n c e n t r a t e d t o a . f i n a l volume o f a p p r o x i m a t e l y 1.0 ml. Each sample was then d i a l y z e d f o r 24 hours and assayed f o r t o t a l p r o t e i n content and u n i t s o f ODC i n h i b i t o r y a c t i v i t y * . A l l assays were done i n duplicate or t r i p l i c a t e .  Eluting Salt Concentration (mM)  *  Total Protein (mg)  Units of Thymus ODC Inhibition*  0  2.38  120  100  1.06  70  300  0.34  1440  500  0.14  0  one u n i t o f i n h i b i t i o n c o r r e s p o n d s t o a r e d u c t i o n o f enzyme a c t i v i t y from c o n t r o l animals by 1 pmole as measured m i x i n g i n a 30 minute assay (see Methods  section).  a l s o a s t r o n g band a t 232,000 d a l t o n s .  DEAE Sepharose CL-6B chromatography to any o t h e r p u r i f i c a t i o n experiments.  was  c a r r i e d out r o u t i n e l y  prior  I t r e s u l t e d i n a p u r i f i c a t i o n of  a p p r o x i m a t e l y 6 f o l d and enabled the c o n c e n t r a t i o n and l o n g term s t o r a g e of the i n h i b i t i n g substance a t 0°C. elsewhere t h a t DEAE Sepharose  In a d d i t i o n ,  i s an e f f e c t i v e procedure f o r near  removal of the d e t e r g e n t L u b r o l ( 8 1 ) . from the e x t r a c t c o n t a i n i n g the ODC sulfate precipitation.  i t has been demonstrated complete  Another method f o r removing  Lubrol  i n h i b i t i n g substance i s ammonium  T h i s procedure was  c a r r i e d out on the crude thymus  e x t r a c t , p r i o r t o i t s a p p l i c a t i o n onto the DEAE Sepharose column. small p u r i f i c a t i o n was  While a  o b t a i n e d , the e x t e n s i v e d i a l y s i s r e q u i r e d to remove  the s a l t r e s u l t e d i n o n l y a 30% r e c o v e r y o f i n h i b i t o r y a c t i v i t y , making i t unsuitable f o r p u r i f i c a t i o n  purposes.  S e v e r a l d i f f e r e n t column chromatography, procedures were i n v e s t i g a t e d f o r use as a d d i t i o n a l s t e p s o f p u r i f i c a t i o n .  Neither gel f i l t r a t i o n ,  c h r o m a t o f o c u s s i n g o r a second i o n exchange column e q u i l i b r a t e d to a h i g h e r pH, r e s u l t e d  i n a workable  system f o r f u r t h e r p u r i f y i n g the i n h i b i t o r .  The use of H e p a r i n Sepharose CL-6B d i d prove p a r t i a l l y s u c c e s s f u l , under c a r e f u l l y c o n t r o l l e d c o n d i t i o n s , the p a r t i a l l y p u r i f i e d i n h i b i t i n g p o l y p e p t i d e d i d b i n d t o the r e s i n . to e l u t e t h i s column as was u s i n g DEAE Sepharose.  results listed  ODC  The same procedure was  developed f o r the i n i t i a l  used  p u r i f i c a t i o n step  The l a r g e m a j o r i t y o f i n h i b i t o r y a c t i v i t y  from the H e p a r i n Sepharose column i n the 100 mM  since  NaCl f r a c t i o n s .  eluted The  i n T a b l e 11 show t h a t a s m a l l amount o f the I n h i b i t i o n d i d  not bind to the column.  P r o t e i n assays were a g a i n c a r r i e d out on  44. TABLE 11 PURIFICATION OF THYMUS ODC INHIBITOR USING HEPARIN SEPHAROSE CL-6B COLUMN CHROMATOGRAPHY T h i s procedure was c a r r i e d out u s i n g t h e p a r t i a l l y p u r i f i e d i n h i b i t o r o b t a i n e d from DEAE Sepharose CL-6B chromatography. The column c o n d i t i o n s are d e s c r i b e d i n the Methods s e c t i o n . The f r a c t i o n s e l u t e d a t each NaCl c o n c e n t r a t i o n were pooled and c o n c e n t r a t e d t o a f i n a l volume o f a p p r o x i m a t e l y 1.0 ml. Each sample was then d i a l y z e d f o r 24 hours and assayed f o r t o t a l p r o t e i n c o n t e n t and u n i t s o f ODC i n h i b i t o r y a c t i v i t y * . A l l assays were done i n d u p l i c a t e o r t r i p l i c a t e .  Eluting Salt Concentration (mM)  *  Total Protein (mg)  Units of Thymus ODC Inhibition*  0  2.1  1180  100  0.7  3890  300  0.5  630  500  0.4  670  one u n i t o f i n h i b i t i o n . c o r r e s p o n d s t o a r e d u c t i o n o f enzyme a c t i v i t y from c o n t r o l animals by 1 pmole i n a 30 minute assay (see Methods section).  i n d i v i d u a l f r a c t i o n s and the r e s u l t s a r e i l l u s t r a t e d s t e p r e s u l t e d i n an average 4 f o l d a d d i t i o n a l  i n Table 11.  This  purification.  These two steps o f p u r i f i c a t i o n were r e p e a t e d s e v e r a l times t o c o n f i r m the r e s u l t and t o accumulate s u f f i c i e n t  q u a n t i t i e s of the  i n h i b i t i n g p o l y p e p t i d e f o r a m o l e c u l a r weight e s t i m a t i o n u s i n g g e l filtration.  A S e p h a c r y l S-200 column  (90 cm x 1.4 cm) was e q u i l i b r a t e d  w i t h s t a n d a r d b u f f e r and c a l i b r a t e d w i t h f i v e m o l e c u l a r weight s t a n d a r d s . The p a r t i a l l y p u r i f i e d  i n h i b i t i n g p o l y p e p t i d e was a p p l i e d t o the column  and the e l u t e d f r a c t i o n s were checked f o r i n h i b i t o r y a c t i v i t y u s i n g the mixing a s s a y .  The r e s u l t s i n F i g u r e 8 a s s i g n an approximate m o l e c u l a r  weight t o t h e ODC i n h i b i t i n g p o l y p e p t i d e o f 54,000 d a l t o n s .  The Role o f P y r i d o x a l Phosphate i n Thymus ODC I n h i b i t i o n P r e l i m i n a r y r e s u l t s suggest t h a t p y r i d o x a l phosphate may be i n v o l v e d i n the mechanism of ODC i n h i b i t i o n i n thymus o f dexamethasone-treated rats.  PAMP A f f i g e l column chromatography  p u r i f y i n g thymus ODC from c o n t r o l r a t s  has proven e f f e c t i v e i n  ( d a t a n o t shown).  When a crude  thymus e x t r a c t from dexamethasone-treated r a t s i s passed through t h i s system, an apparent r e g e n e r a t i o n o f ODC a c t i v i t y i s d e t e c t e d a t the v o i d volume.  When t h e f r a c t i o n s a r e assayed f o r i n h i b i t o r y a c t i v i t y , a s m a l l  peak i s d e t e c t e d near t h e same p o s i t i o n i n t h e column e l u t e d when p u r i f i e d from c o n t r o l a n i m a l s .  p r o f i l e a t which ODC  Mixing assays using  extracts  from r a t thymus and thymus o f dexamethasone-treated r a t s were conducted a t v a r i o u s PLP c o n c e n t r a t i o n s .  These r e s u l t s a r e i n c o n c l u s i v e s i n c e t h e  a c t i v i t y of thymus ODC i n c o n t r o l r a t s i s markedly reduced i n the absence of PLP.  However, t a k i n g t h a t i n t o a c c o u n t , t h e i n h i b i t i o n measured i n  46.  FIGURE 8 MOLECULAR WEIGHT DETERMINATION OF THYMUS ODC INHIBITOR M o l e c u l a r weight s t a n d a r d s were used t o c a l i b r a t e a S e p h a c r y l S-200 g e l f i l t r a t i o n column. A p a r t i a l l y p u r i f i e d thymus ODC i n h i b i t o r e x t r a c t was a p p l i e d t o the column and i t was e l u t e d from t h e column a t a p o i n t c o r r e s p o n d i n g t o a m o l e c u l a r weight o f 54,000 d a l t o n s . T h i s procedure i s d i s c u s s e d i n d e t a i l i n the Methods s e c t i o n . The s t a n d a r d s used were: Blue d e x t r a n (2 x 1 0 ) , F e r r i t i n (440,000), C a t a l a s e (332,000), A l d o l a s e (158,000) and B.S.A. (67,000). 6  (J\A/) * H ! M B  9  JD|nDa|oyy^  47. the absence o f PLP i s s t i l l  h i g h e r than t h a t w i t h PLP p r e s e n t .  Perhaps a  c l e a r e r r e s u l t would be o b t a i n e d i f t h e m i x i n g assays were c a r r i e d out using p a r t i a l l y p u r i f i e d  i n h i b i t o r and enzyme.  48. DISCUSSION  The  r e s u l t s reported  i n thymus and  spleen  here suggest t h a t the decrease i n ODC  of dexamethasone-treated r a t s i s r e l a t e d to  presence of an i n h i b i t o r of ODC treatment.  The  spleen  s i g n i f i c a n t l y enhanced by L u b r o l .  v a r i a t i o n i n i n h i b i t o r y a c t i v i t y was e x t r a c t e d w i t h Hepes b u f f e r o n l y .  C o n s i s t e n t l y high  combined w i t h the aqueous e x t r a c t .  A wide  l e v e l s of  inhibition  1% L u b r o l ,  e x t r a c t s d i d not  t o t a l protein concentrations  p o s s i b l e t h a t the d e t e r g e n t h e l p e d break up  w i t h the motor d r i v e n homogenizer a l o n e . from c o n t r o l r a t s was as low  d e t e r g e n t b r i n g i n g about the  extracted to i n h i b i t  The  of the a s s a y c o n d i t i o n s  i n h i b i t o r y a c t i v i t y was  detected  f o r a second time u s i n g  accomplished  a c t i v i t y of ODC  T h i s may  i n crude  by  r e s u l t from  the  when the  i n d i c a t i n g t h a t i n h i b i t i o n of ODC  present  o b s e r v e d i n h i b i t i o n of ODC  i s not  or the e x t r a c t i o n procedure.  when the  thymus of c o n t r o l r a t s  1% L u b r o l and  enzyme from c o n t r o l r a t s .  shown to remain c o n s t a n t  be  r e l e a s e of p r o t e o l y t i c enzymes, not  i n the thymus, which degrade ODC. an a r t i f a c t  The  markedly reduced  as 0.1%.  It i s  the c e l l s i n a more e f f i c i e n t  manner to produce a c o n s i s t e n t homogenate which c o u l d not  of L u b r o l  in  r i s e s i g n i f i c a n t l y a f t e r a second  L u b r o l e x t r a c t i o n as compared w i t h a second b u f f e r e x t r a c t i o n .  concentrations  was  T h i s e f f e c t does not appear to r e s u l t  from the r e l e a s e of a d d i t i o n a l p r o t e i n as  e x t r a c t s of s p l e e n  of  observed when the t i s s u e s were  were observed when a second e x t r a c t i o n , c a r r i e d out u s i n g  spleen  the  produced i n response to the hormone  e x t r a c t i o n of the i n h i b i t o r from thymus and  dexamethasone-treated r a t s was  thymus and  activity  Also,  substrate  No was  then assayed f o r a b i l i t y  the degree of i n h i b i t i o n concentration  a c t i v i t y was  not  due  to  was  varied,  limiting  was  49.  c o n c e n t r a t i o n s of s u b s t r a t e .  The i n h i b i t o r appears t o be a p o l y p e p t i d e , s i n c e i t s a c t i v i t y destroyed by treatment w i t h heat or w i t h t r y p s i n .  was  Some s p e c i f i c i t y of the  i n h i b i t o r i s suggested, s i n c e experiments showed t h a t i n h i b i t i o n of SAMD d i d not occur i n the m i x i n g a s s a y s .  Since i t r e t a i n s i t s i n h i b i t o r y  f u n c t i o n a f t e r d i a l y s i s and a f t e r u l t r a f i l t r a t i o n w i t h e x c l u s i o n l i m i t s of up to 50,000 d a l t o n s , the ODC  i n h i b i t o r i n d u c e d i n r a t thymus by  dexamethasone i s u n l i k e l y t o r e q u i r e a s m a l l m o l e c u l e f o r a c t i v i t y . would seem t o d i f f e r e n t i a t e i t s a c t i o n from the group of ODC that f u n c t i o n through p o s t - t r a n s l a t i o n a l m o d i f i c a t i o n .  This  inhibitors  Both the  t r a n s g l u t a m i n a s e (51) which r e q u i r e s p u t r e s c i n e , and the p r o t e i n k i n a s e 2+ (53) which r e q u i r e s Mg extensive d i a l y s i s .  /ATP  Low  and a polyamine, would be i n a c t i v a t e d  l e v e l s of i n h i b i t i o n a r e d e t e c t e d at 2.5  by hours  a f t e r dexamethasone i s a d m i n i s t e r e d , w h i l e h i g h i n h i b i t o r y a c t i v i t y i s seen a t 5 and 12 hours a f t e r the r a t s r e c e i v e d i s measurable  the hormone.  24 hours a f t e r dexamethasone t r e a t m e n t .  suggest t h a t i n the e a r l i e s t most of the i n h i b i t o r may  These  No  inhibition  results  s t a g e s o f the hormone induced l y t i c  be complexed  response,  w i t h enzyme and a t a l a t e r  time,  excess i n h i b i t o r i s p r e s e n t and t h e r e f o r e d e t e c t a b l e by a mixing assay. The absence  of i n h i b i t o r y a c t i v i t y a t the l a t e r s t a g e s of the t i s s u e  response i n d i c a t e s t h a t the l a r g e m a j o r i t y of the lymphocytes  had been  destroyed.  I t has been e s t a b l i s h e d t h a t the ODC dexamethasone-treated  i n h i b i t o r i n thymus of  r a t s i n t e r a c t s w i t h the enzyme i n such a way  a l t e r i t s chromatographic  properties (77).  as t o  The mechanism of i n h i b i t i o n  50.  appears  to be n o n - c o m p e t i t i v e and n o n - c a t a l y t i c .  ornithine  were determined  f o r ODC  i n thymus o f c o n t r o l  molecules i n mixing a s s s a y s of c o n t r o l the degree of i n h i b i t i o n v a r i e s  Identical  stoichiometrically  Thus, i f the i n h i b i t o r i n t e r a c t s  so i n a n o n - c a t a l y t i c manner, p o s s i b l y  r a t s and a c t i v e  enzyme and i n h i b i t o r .  volumes of i n h i b i t o r e x t r a c t and enzyme e x t r a c t , time.  Km v a l u e s f o r ODC  Also, while  w i t h the r a t i o of  i t does not v a r y w i t h  w i t h the enzyme d i r e c t l y , i t does to form an  inactive  e n z y m e - i n h i b i t o r complex.  The procedure used t o p a r t i a l l y p u r i f y e x t r a c t s of dexamethasone-treated steps.  rats  further  m o l e c u l a r weight  purification.  substantially Sepharose above 35%.  i n v o l v e d two column chromatography  The  d e t e r m i n a t i o n , i t d i d not r e s u l t  eluting  h i g h e r i f the r e c o v e r y r a t e s a s s o c i a t e d w i t h both the DEAE  and H e p a r i n Sepharose I t was  column chromatography procedures had been  found t h a t a p p r o x i m a t e l y 20% of the i n h i b i t o r y a c t i v i t y resin.  In a d d i t i o n ,  the method of  both columns d i d not o p t i m i z e r e c o v e r y , s i n c e s i g n i f i c a n t  the i n h i b i t o r o c c u r r e d .  individual  fractions  not p r a c t i c a l  since  would then have t o undergo d i a l y s i s t o remove s a l t  to assay f o r ODC  and i n h i b i t o r a c t i v i t y .  The m o l e c u l a r weight determined  dilution  A g r a d i e n t o f i o n i c s t r e n g t h f o r column  e l u t i o n would have been p r e f e r a b l e , but t h i s was  mM  i n any  o v e r a l l p u r i f i c a t i o n o f 24 f o l d would be  d i d not b i n d t o the H e p a r i n Sepharose  prior  i n h i b i t o r i n thymus  While the S e p h a c r y l S-200 g e l f i l t r a t i o n procedure gave an  approximate  of  the ODC  o f the p a r t i a l l y p u r i f i e d  to'be a p p r o x i m a t e l y 54,000 a t pH 7.4  d i t h i o t h r e i t o l and 100 mM  EDTA.  This finding  inhibitor  i n 50 mM  was  Hepes b u f f e r ,  again d i f e r e n t i a t e s  10 the  51.  thymus ODC i n h i b i t o r from o t h e r r e p o r t e d ODC i n h i b i t o r s .  The ODC  i n a c t i v a t i n g substance found i n r a t p r o s t a t e (57) and the ODC found i n many t i s s u e s  antizyme  (44) have m o l e c u l a r weights o f 26,000 o r l e s s .  The  ODC i n h i b i t o r found i n r a t thymus seems t o resemble a substance found i n slow growing  r a t p r o s t a t i c tumor ( 8 0 ) .  but has not y e t been f u l l y  T h i s i n h i b i t o r had a Mr of 68,000,  characterized.  While i n h i b i t o r s of ODC have been r e p o r t e d by o t h e r l a b o r a t o r i e s , u s i n g o t h e r b i o l o g i c a l systems,  t h i s i s the f i r s t  i n s t a n c e i n which the  o c c u r r e n c e o f an i n h i b i t o r has been observed i n response to a p h y s i o l o g i c a l s t i m u l u s w i t h a d e f i n e d l o n g range e f f e c t .  Similar  effects  of d i b u t y r y l cAMP and dexamethasone on the d e c a r b o x y l a s e s have been found i n c u l t u r e d S49 lymphoma c e l l s  (63).  However, an i n h i b i t o r of ODC was not  found i n an e x t r a c t o f d i b u t y r y l cAMP i n h i b i t e d c e l l s on mixing w i t h extracts of control c e l l s  (81).  The r e s u l t s r e p o r t e d here f o r thymus and  s p l e e n a i d i n the u n d e r s t a n d i n g of the r o l e o f g l u c o c o r t i c o i d s i n r e g u l a t i n g growth i n l y m p h a t i c t i s s u e .  I t i s a l r e a d y known that the  n e g a t i v e growth response i n d u c e d i n thymus by g l u c o c o r t i c o i d s  requires  p r o t e i n s y n t h e s i s and t h a t a major s p e c i e s o f ODC a c t i v i t y i s absent  soon  a f t e r hormone a d m i n i s t r a t i o n and w e l l b e f o r e t h e c e s s a t i o n of c e l l d i v i s i o n and e v e n t u a l c y t o l y s i s . study appears  The ODC i n h i b i t o r i d e n t i f i e d i n t h i s  to p l a y a l a r g e r o l e i n m e d i a t i n g t h i s  response.  52.  BIBLIOGRAPHY  1.  Thomas, N. and B e l l , P.A (1981)  M o l e c . and C e l l . Endocr. _22, 71-84.  2.  Pegg, A.E., S e e l y , J . E . , Pb'so, H., Ragione, F.D. and Zagon, I.S. (1982) F e d . P r o c . 41, 3065-3072.  3.  Vete, T. (1966)  4.  S c a l a b r i n o , G., F e r i o l i , M.E., B a s a q u i , M., N e b u l o r i , R. and F r a s c h i n i , F. (1979) Amer. J . P h y s i o l . 237, ( 1 ) : E6-E10.  5.  Friedman, Y., Park, S., L e v a s s e u r , S. and Burke, G. (1977) B i o p h y s . A c t a 500, 291-303.  6.  E r w i n , B.G., S e e l y , J . E . and Pegg, A.E. (1983) 3027-3032.  7.  S e e l y , J . E . , Pbsb, H. and Pegg, A.E. (1982) 13, 7549-7553.  8.  R i c h a r d s , J . F . (1978)  9.  Ganem, B. (1982)  B i o c h i m . B i o p h y s . A c t a 121, 386-394.  Life  Biochim.  B i o c h e m i s t r y 22,  J . B i o l . Chem. 257, No.  S c i e n c e s _23, 1619-1624.  A c c . Chem. Res. 15, 290-298.  10.  Isomaa, V.V., Pajunen, A . E . I . , B a r d i n , C.W. J . B i o l . Chem. 258, No. 11, 6735-6740.  and Janne, O.A. (1983)  11.  R u s s e l , D.H., Snyder, S.H. and Medina, V . J . (1970) 1414-1419.  12.  Brandt, J.T., P i e r c e , D.A. and F a u s t o , N. (1972) A c t a 279, 184-193.  13.  P e r r s o n , L . (1981)  14.  K i t a n i , T. and F u j i s a w a , II. (1983) 235-239.  15.  Ono, M., Inove, H., S u z i k i , F. and Takeda, Y. (1972) B i o p h y s . A c t a 284, 285-297.  16.  Haddox, M.K. and R u s s e l , D.H. (1981)  17.  Janne, J . and Williams-Ashman, H.G. (1971) 1725-1732.  18.  S e e l y , J . E . , Posb, H. and Pegg, A.E. (1982) 3394-3399.  19.  JSnne, J . and Williams-Ashman, H.G. (1970)  E n d o c r i n o l o g y 86,  Biochim. Biophys.  A c t a . Chem. Scand. Sev. B35, 737-738. J . B i o l . Chem. 258, NI. 1,  Biochim.  B i o c h e m i s t r y , _20, 6721-6729. J . B i o l . Chem. 246,  B i o c h e m i s t r y 21,  Biochem. J . 119, 595-597.  53.  20.  Boucek, R.J. and Lembach, K. (1977) 408-415.  A r c h . Biochem. Biophys. 184,  21.  Weiss, J.M., Lembach, K. and Boucek, R.J. (1981) 229-239.  22.  Pegg, A.E. and M c G i l l , S. (1979)  23.  K i t a n i , T. and F u j i s a w a , H. -(1981)  24.  T y a g i , A.K., Tabor, C.W. and Tabor, H. (1981) 12156-12163.  25.  Friedman, S.J., H a l p e r n , K.V. and C a n e l l a k i s , E.S. (1972) Biophys. A c t a 261, 181-187.  26.  Polyamines i n B i o l o g y and M e d i c i n e (1981) ed. M o r r i s , D.R. and Marton, L . J . , V o l . 8. The B i o c h e m i s t r y o f D i s e a s e , M. Dekker, New York.  27.  Obenrader, M.F. and P r o u t y , W.F. (1977) 2860-2865.  28.  P r o u t y , W.F. (1976)  29.  H o l t t a , E., Janne, J . and P i s p a , J . (1972) Comm. 47, 1165-1171.  30.  M i t c h e l l , J.L.A., C a r t e r , D.D. and R y b s k i , J.A. (1978) Biochem. 92_, 325-331.  31.  Raina, A. and Janne, J . (1968)  32.  Williams-Ashman, H.G. and C a n e l l a k i s , Z.N. (1979) B i o l , and Med. 22, 421.  33.  R u s s e l , D.H. (1981)  34.  Janne, J . and Raina, A. (1968)  35.  Pegg, A.E. and Williams-Ashman, H.G. (1968) Biochem. J . 108, 433-539.  36.  M o r r i s , D.R. and F i l l i n g a m e , R.H. (1974) 43_, 303-325.  37.  Tabor, H. and Tabor, C.W. (1983) A d . i n Polyamine Research, V o l . 4, p. 455-463, ed. V. Bachrach, A. Kaye and R. Chayen^ Raven P r e s s , N.Y.  Biochem. J . 194,  B i o c h i m . Biophys. A c t a 568, 416-427. E u r . J . Biochem. 119, 177-181. J . B i o l . Chem. 256,  Biochim.  J . B i o l . Chem. 252,  J . C e l l . P h y s i o l . _89, 65-76.  Med. B i o l .  Biochem. Biophys. Res.  Eur. J .  A c t a . Chem. Scand. 22., 2375-2378. Perspectives i n  59_, 286-295. A c t a . Chem. Scand. 22., 1349-1351.  Ann. Rev. o f B i o c h e m i s t r y  38. • R a i n a , A., E l o r a n t a , T., Hyvbnen, T. and P a j u l a , R.L. (1983) Polyamine Research V o l . 4. 39.  F i l l i n g a m e , R.H. and M o r r i s , D.R. Comm. 52, 1020-1025.  (1973)  Ad. i n  Biochem. Biophys. Res.  54.  40.  C i d l o w s k i , J.A. and C i d l o w s k i , N.B. (1981) " C a n c e r Research 41, 2687-2691.  41.  Pegg, A.E., Tang, K.C. and Coward, J.K. (1982) 5089-5096.  42.  S e e l y , J . E . and Pegg, A.E. (1983)  43.  H e l l e r , J.S., Fong, W.F. and C a n e l l a k i s , E.S. (1976) 1858-1862.  44.  K y r i a k i d i s , D.A., F l a m i g n i , F., Pawlak, J.W. and C a n e l l a k i s , E.S. (1984) Biochem. Pharm. 33_, 1575-1578.  45.  H e l l e r , J.S., Fong, W.F. and C a n e l l a k i s , E.S. (1977) Biochem. 81_, 545-550.  46.  H e l l e r , J.S., Chen, K.Y., K y r i a k i d i s , D.A., Fong, W.F. and C a n e l l a k i s , E.S. (1978) J . C e l l P h y s i o l . 96_, 225-236.  47.  McCann, P.P., T a r d i f , C. and Mamont, P.S. (1977) Res. Comm. 7_5, 948-954.  48.  F u j i t a , K., Murakami, Y. and H a y a s h i , S - I . (1982) 647-652.  49.  G r i l l o , M.A., Bedino, S. and T e s t o v e , G. (1980) 37-42.  50.  L e s i e w i c z , J . and G o l d s m i t h , L.A. (1983)  51.  R u s s e l , D.A. (1981)  52.  Haddox, M.H. and R u s s e l , D.H. (1981)  53.  Atmar, V . J . and Kuehn, G.D. (1981)  P.N.A.S. 78_, 5518-5522.  54.  Kuehn, G.D. and Atmar, V . J . (1982)  F e d . P r o c . 4T, 3078-3083.  55.  Atmar, V . J . , Kuehn, G.D. and C a s i l l a s , E.R. (1981) 256, 8275-8278.  56.  Kuehn, G.D. and Atmar, V . J . (1983)  57.  I c e k s o n , I . and Kaye, A.M. (1976)  58.  Kaye, A.M. and Y a r i v , M. (1983)  59.  A t k i n s , F.L. and Beaven, M.A. (1975)  60.  Beaven, M.A. and De Jong, W. (1972)  B i o c h e m i s t r y 21,  J . B i o l . Chem. 258, 2496-2500. P.N.A.S. 73,  Eur. J .  Biochem. Biophys.  Biochem. J . 204,  I n t . J . Biochem. 11,  J . I n v e s . Derm. 80, 97-100.  Biochem. B i o p h y s . Res. Comm. 99_, 1167-1172. J . C e l l . P h y s i o l . 109, 447-452.  J . B i o l . Chem.  Ad. Polyamine Research 4_, 615-629. FEBS L e t t . 61_, 54-58.  Ad. Polyamine Research 4_, 693-703. Biochem. Pharm. 24_, 763-768. Biochem. Pharm. 22, 257-265.  55.  61.  Guyton, A.D. (1976) Saunders, T o r o n t o .  62.  Frank, L . and R o b e r t s , R. (1979)  63.  Honeysett, J.M. and I n s e l , P.A. 321-332.  64.  McConlogue, L.C., Marton, L . J . and C o f f i n o , P. (1983) 96_, 762-767.  65.  McConlogue, L.C. and C o f f i n o , P. (1983) 12083-12086.  66.  Kay, J . E . and L i n d s a y , V . J . (1973)  Biochem. J . 132,  67.  Kay, J . E . and L i n d s a y , V . J . (1973)  E x p t . C e l l Res. 77_, 428.  68.  R u s s e l , D.A.  69.  L e h n i n g e r , A.L. (1975)  70.  Janne, J . , P'dso, H. and Raina, A. (1978) 241-293.  71.  R i c h a r d s , J . F . , L i t , K., Fuca, R. and B o u r g e a l t , C. (1981) Biophys. Res. Comm. 99_, 14461-1467.  72.  Noguchi, T., K a s h i w a g i , A. and Tanaka, T. (1976) 451-454.  73.  I s o h u s h i , F., Tsukanaka, K., Tevada, M., N a k a n i s h i , Y., Tami, S. and Sakamoto, Y. (1980) Can. Res. 40_, 877-881.  74.  Harmon, J.M., Norman, M.R., Fowlkes, B . J . and Thompson, B.E. (1979) J . C e l l . P h y s i o l . 98_, 267-278.  75.  N i c h o l s o n , M.L., 3530-3537.  (1980)  Textbook of M e d i c a l P h y s i o l o g y 4th E d i t i o n ,  B i o l . Neonate 36^, 1-9. (1981)  Pharmacology 20,  J. Cyclic  Nuc. Res. ]_,  Biol.  791-796.  117-129.  B i o c h e m i s t r y , 2nd Ed., Worth, New York. Biochem. Biophys. A c t a 473,  (1981)  J.M. and B o u t w e l l , R.K.  Biochim.  J . Biochem. 79,  Cancer Res. 41,  (1983)  Sci.  Tl  77.  Thomson, M.J. and R i c h a r d s , J . F . (1978)  Life  78.  N a r v a t a , H., Yamamoto, R.S. and P o i r i e r , 689-698.  L.A. (1978)  79.  B r a d f o r d , M. (1976)  80.  Olson, J.W.  81.  I n s e l , P.A. and Fenno, J . (1978)  (1982)  J. Cell  J . B i o l . Chem,. 258,  V o r i s , B.P. and Young, D.A.  76.. A s h e n d e l , C.L., S t a l l e r , 43_, 4327-4332.  W.B.  Cancer Res.  337-344.  y  Life  A n a l . Biochem. _72, 248-254. P r o c . Amer. A s s o c . Cane. Res. 23_, #60. P.N.A.S. 75, 862-865.  Sci.  26,  

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