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Specificity and kinetic studies of deoxyribonucleases from the intestinal mucosa of the rat Lee, Cheuk Yu 1968

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SPECIFICITY AND KINETIC STUDIES OF DEOXYRIBONUCLEASES FROM THE INTESTINAL MUCOSA OF THE RAT by CHEUK YU LEE B.Sc,  U n i v e r s i t y o f B r i t i s h Columbia, 1963  M.Sc,  U n i v e r s i t y o f B r i t i s h Columbia, 1965  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE  REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  i n the Department of BIOCHEMISTRY  We accept t h i s t h e s i s as conforming to the required  THE  standard  UNIVERSITY OF BRITISH COLUMBIA May, 1 9 6 8  In p r e s e n t i n g t h i s  thesis  advanced  the  Library  degree  at  s h a l l make  agree that  it  University freely  tatives.  It  financial  gain  of  / " ^ y  requirements  for  Columbia,  I agree that  the  reference  copying of  this  not  and s t u d y . thesis  o r by h i s  represen-  (J^c?-  this  thesis  permission.  an  further  scholarly  be a l l o w e d w i t h o u t my w r i t t e n  Columbia  I  for  copying o r p u b l i c a t i o n of  Biochemistry  ^  for  the  by t h e Head o f my D e p a r t m e n t  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8. C a n a d a Date  available  is understood that  shall  f u l f i l m e n t of  of B r i t i s h  permission for extensive  p u r p o s e s may be g r a n t e d  Department  in p a r t i a l  for  ii ABSTRACT The p r o p e r t i e s o f the deoxyribonuclease a c t i v i t y i n the i n t e s t i n a l mucosa o f the r a t have been s t u d i e d .  Two DNases  were found i n a c e l l - f r e e e x t r a c t prepared by homogenizing mucosal t i s s u e i n Krebs-Ringer phosphate  the  b u f f e r and then cen-  t r i f u g i n g the homogenate a t 1 0 5 , 0 0 0 x g f o r 60 mins.  Resolu-  t i o n and p a r t i a l p u r i f i c a t i o n of these two enzymes were a c h i e v e d by ion-exchange  chromatography on D E A E - c e l l u l o s e and p a r t i t i o n  on h y d r o x y l a p a t i t e of the c e l l - f r e e e x t r a c t o r an acetone powder p r e p a r a t i o n o f the enzymes. One o f the enzymes was i d e n t i f i e d as DNase I by i t s optimum pH ( 6 . 5 to 6 . 8 ) , i t s requirements f o r b i v a l e n t  metals  and by i t s r e a c t i o n to known DNase I i n h i b i t o r s such as EDTA, clfcrate and a r s e n a t e .  I t was a l s o found to be a c t i v e  toward  n a t i v e DNA,and, to a l e s s e r degree, toward heated DNA. second DNase was shown to be q u a l i t a t i v e l y d i f f e r e n t DNase I .  I t has an a c i d i c optimum pH ( 3 . 5 - ^ ^ ) ,  The  from  does not r e -  q u i r e a c t i v a t i o n by b i v a l e n t c a t i o n s and i s not i n h i b i t e d by EDTA, c i t r a t e o r a r s e n a t e .  T h i s second DNase a c t i v i t y i s  t h e r e f o r e o f the DNase I I type. The l i n k a g e s p e c i f i c i t y o f the two enzymes was by i s o l a t i n g the p r o d u c t s o f the r e a c t i o n and examining  studied them  with r e s p e c t t o c h a i n l e n g t h , base composition o f the mononuc l e o t i d e s , r e l a t i v e f r e q u e n c i e s o f the d i n u c l e o t i d e s and  iii base f r e q u e n c i e s a t the ends o f the o l i g o n u c l e o t i d e s . The h y d r o l y s i s r e a c t i o n was c a r r i e d out under a v a r i e t y o f conditions.  When M g  was used as the a c t i v a t i n g i o n and n a t i v e DNA  + +  as s u b s t r a t e , DNase I was found to show a p r e f e r e n c e l i n k a g e s pApC, pApT and pGpT.  But when M n  ++  f o r the  was the a c t i v a t o r ,  o r when heated DNA was used as s u b s t r a t e i n the presence o f M g DNase I d i d not show any s i g n i f i c a n t order o f s p e c i f i c i t y .  ++  With  r e g a r d t o DNase I I , the enzyme was found to a t t a c k n a t i v e DNA p r e f e r e n t i a l l y a t the ApCp, GpGp and GpTp bonds. The  mechanism o f metal a c t i v a t i o n o f i n t e s t i n a l  DNase I was a l s o s t u d i e d .  P r e l i m i n a r y experiments showed that  the enzyme was i n h i b i t e d by h i g h c o n c e n t r a t i o n s o f both metals and DNA.  I n a d d i t i o n , o p t i m a l a c t i v a t i o n was found to depend  on the molar r a t i o o f metal i o n t o DNA phosphorus. riments  These expe-  suggested t h a t the metal-DNA complex i s probably the  true substrate.  Consequently, a r a t e equation  i n terms o f the  metallosubstrate  c o n c e n t r a t i o n was developed based on the  assumption t h a t i n t e s t i n a l DNase I can combine i n d i v i d u a l l y and  simultaneously  complex.  with f r e e metal,  Data o b t a i n e d  f r e e DNA and the metal-DNA  from i n i t i a l v e l o c i t y s t u d i e s c a r r i e d  out a t v a r y i n g c o n c e n t r a t i o n s o f metal and DNA f i t t e d r a t e equation w e l l .  this  On t h i s b a s i s , i t was suggested t h a t the  enzyme, the metal a c t i v a t o r and the DNA s u b s t r a t e combine to form a t e r n a r y complex which then d i s s o c i a t e s to g i v e the products.  iv TABLE OF CONTENTS Page 1  INTRODUCTION ISOLATION AND PURIFICATION  OF DEOXYRI BO NUCLEASES . .  EXPERIMENTAL 1.  18  Enzyme Assays  18  2. P r e p a r a t i o n of Crude I n t e s t i n a l E x t r a c t  . . .  3. P r e p a r a t i o n o f Acetone Powder 4.  18  D e s a l t i n g w i t h Sephadex G-25  19 20 21  Columns  5. Ion-Exchange Chromatography  21  6. Hydroxylapat jte Chromatography  2^1-  7.  25  L i g a t i n g o f P a n c r e a t i c Duct o f Rat  RESULTS AND  26  DISCUSSION  SPECIFICITY OF THE RAT INTESTINAL  DEOXYRI BO NUCLEASES  35  MATERIALS  35  EXPERIMENTAL  35  1.  P r e p a r a t i o n o f DNA H y d r o l y s a t e  35  2.  Ion-Exchange Chromatography o f DNA Hydrolysate 36  3. Removal o f Urea  38  4. Determination o f Chain Length of O l i g o ^0  nucleotides 5. Base Composition o f Mononucleotide F r a c t i o n  .  6. End-Group A n a l y s i s 7.  S e p a r a t i o n and A n a l y s i s o f D i n u c l e o t i d e s  8. Denaturation o f C a l f Thymus DNA  4-1 k-2  . .  ^3 4-8  V  Page 9. S h e a r i n g o f C a l f Thymus DNA  48  RESULTS AND DISCUSSION  48  KINETIC STUDIES OF THE INTESTINAL DEOXYRIBONUCLEASE I REACTION EXPERIMENTAL 1.  Measurement o f DNase A c t i v i t y  "  70 70 70  2. M e t a l B i n d i n g S t u d i e s  71  3. O p t i c a l Rotary D i s p e r s i o n Studies  72  RESULTS AND DISCUSSION 1.  73  E f f e c t of Substrate Concentration  76  2. E f f e c t o f M e t a l I o n C o n c e n t r a t i o n  79  3.  84  Further Studies of Substrate I n h i b i t i o n . . . .  4. D e t e r m i n a t i o n o f K i n e t i c Parameters  87  5. D e r i v a t i o n o f Rate E q u a t i o n  100  6. I n h i b i t i o n S t u d i e s  123  SUMMARY  127  REFERENCES  130  vi LIST OF TABLES Table  Page  I.. P u r i f i c a t i o n o f I n t e s t i n a l DNases  32  I I . B u f f e r S o l u t i o n s Used i n Chromatography of D i n u c l e o t i d e s  45  I I I . Percentage Base C o m p o s i t i o n o f M o n o n u c l e o t i d e s IV.  Percentage Frequencies o f D i n u c l e o t i d e s . . . .  55 56  V. P e r c e n t a g e Base F r e q u e n c i e s a t t h e Ends of T r i n u c l e o t i d e s  57  V I . P e r c e n t a g e Base F r e q u e n c i e s a t t h e Ends of O l i g o n u c l e o t i d e s  58  V I I . Percentage o f O l i g o n u c l e o t i d e Phosphorus as Compared t o T o t a l  6l  V I I I . V a l u e s o f v, £, l / y and l/£ a t Two Concentrations of Total Mg  85  + +  IX. I n i t i a l V e l o c i t i e s o f t h e M n - a c t i v a t e d + +  I n t e s t i n a l DNase I R e a c t i o n  89  X. I n i t i a l V e l o c i t i e s o f t h e M g - a c t i v a t e d + +  I n t e s t i n a l DNase I R e a c t i o n XI.  90  Molar C o n c e n t r a t i o n o f t h e Mn-DNA Complex a t V a r i o u s C o n c e n t r a t i o n s o f DNA and M n  + +  ...  91  X I I . Molar C o n c e n t r a t i o n o f t h e Mg-DNA Complex a t ++ V a r i o u s C o n c e n t r a t i o n s o f DNA and Mg  ...  X I I I . Table o f V a l u e s o f 1/v, l/g and a/v f o r t h e M a n g a n e s e - a c t i v a t e d DNase I R e a c t i o n .  92  vii Table  Page  XIV. Summary o f K i n e t i c Parameters o f the M n - a c t i v a t e d DNase I R e a c t i o n + +  107  XV. Summary o f K i n e t i c Parameters o f the M g - a c t i v a t e d DNase I R e a c t i o n + +  108  viii LIST OF FIGURES Figure  Page  1. Ion-Exchange Chromatography o f C e l l - F r e e E x t r a c t o f I n t e s t i n a l Mucosa o f Rat  27  2. Ion-Exchange Chromatography o f Acetone Powder P r e p a r a t i o n o f I n t e s t i n a l DNases . . . .  28  3. Chromatography o f Rat I n t e s t i n a l DNase I on H y d r o x y l & p a t i t e  30  4. Chromatography o f Rat I n t e s t i n a l DNase I I on H y d r o x y l a p a t i t e  31  5. Chromatography o f DNA H y d r o l y s a t e 6. Ion-Exchange Chromatography o f D i n u c l e o t i d e s  39 .  7. M e l t i n g P r o f i l e s o f V a r i o u s l y - T r e a t e d DNA . . .  46 50  8. E f f e c t o f S u b s t r a t e C o n c e n t r a t i o n o f t h e I n t e s t i n a l DNase I R e a c t i o n 9. E f f e c t o f M g  + +  77  C o n c e n t r a t i o n on t h e I n t e s t i n a l  DNase I R e a c t i o n 10. E f f e c t o f M n  + +  80  Concentration o f the I n t e s t i n a l  DNase I R e a c t i o n  82  11. Lineweaver-Burk P l o t o f t h e M g - a c t i v a t e d + +  I n t e s t i n a l DNase I R e a c t i o n 12. K i n e t i c s  86  o f t h e M n - a c t i v a t e d DNase I + +  Reaction  94  13. Lineweaver-Burk P l o t o f t h e M n - a c t i v a t e d + +  I n t e s t i n a l DNase I R e a c t i o n  95  ix Figure  Page  14. Lineweaver-Burk P l o t o f the M n - a c t i v a t e d + +  96  I n t e s t i n a l DNase I R e a c t i o n 1 5 . Hanes P l o t of the M n - a c t i v a t e d  Intestinal  + +  97  DNase R e a c t i o n 16.  Lineweaver-Burk P l o t o f the M g ^ - a c t i v a t e d 98  I n t e s t i n a l DNase I R e a c t i o n 17.  Plot of V Free M n  and K  a p p  a p p  vs. Concentration of 109  f o r the DNase I R e a c t i o n  ++  18. P l o t o f 1 / V  a p p  v s . 1/a f o r the Mn++-activated 110  I n t e s t i n a l DNase I R e a c t i o n 19.  Plot of 1 / K  a p p  v s . 1/a f o r the M n - a c t i v a t e d + +  I n t e s t i n a l DNase I R e a c t i o n 20.  Plot of a / K 2  a p p  .I l l  v s . a f o r the M n - a c t i v a t e d + +  112  I n t e s t i n a l DNase I R e a c t i o n 21.  Plot of Free M g  and K  a p p  vs. Concentration of  f o r the DNase I R e a c t i o n  ++  2 2 . Plot of l / V ^ 3  1 5  v s . 1/a f o r the M g - a c t i v a t e d + +  I n t e s t i n a l DNase I R e a c t i o n 23. Plot of 1/K  a p p  114  v s . 1/a f o r the M g - a c t i v a t e d + +  115  I n t e s t i n a l DNase I R e a c t i o n 24. P l o t o f a / K 2  a p p  v s . a f o r the M g - a c t i v a t e d + +  I n t e s t i n a l DNase I R e a c t i o n 25.  113  116  E f f e c t o f EDTA, C i t r a t e and Arsenate on the I n t e s t i n a l DNase I R e a c t i o n  125  X  ACKNOWLEDGMENT The  author wishes to express h i s  sincere  g r a t i t u d e to Dr. S.H. Zbarsky f o r h i s guidance, encouragement and c o n s t r u c t i v e c r i t i c i s m during the  course o f t h i s work. Thanks a r e a l s o due Miss Ruth K r e h b i e l f o r  c a r r y i n g out the s p e c t r o p o l a r i m e t r y and  experiments  Mr. L o u i s Neering f o r making the c o n d u c t i v i t y  measurement s. The  support o f the N a t i o n a l Research C o u n c i l  i n the form o f a studentship ledged.  i s g r a t e f u l l y acknow-  xi ABBREVIATIONS A  Adenine  C  Cytosine  G  Guanine  T  Thymine  X  Any Base  Pu  Purine  Py  P y r i m i d i n e base  DNA  Deoxyribonucleic  RNA  Ribonucleic acid  t-RNA •  T r a n s f e r RNA  poly-dC, e t c .  Polymer o f d e o x y c y t i d y l i c a c i d , e t c .  pPupPy  D i n u c l e o t i d e formed by one purine and one p y r i m i d i n e d e o x y r i b o t i d e ; a l s o denotes and i n t e r n u c l e o t i d e bond between one purine and one p y r i m i d i n e deoxyriboside  DNase  Deoxyribonuclease  260  Absorbance a t 260 mu u s i n g a 1 cm l i g h t path cuvette  A  base  acid  DEAE-  Diethylaminoethyl-  EDTA  Ethylenediaminetetraacetate  -1-  INTRODUCTION Terminology  and  Classification  Deoxyribonucleases  (DNases) are a subgroup o f phos-  p h o d i e s t e r a s e s , a g e n e r i c name g i v e n to a group o f enzymes capable of h y d r o l y s i n g d i e s t e r i f i e d phosphate bonds. are endonucleases,  DNases  i . e . , they a t t a c k the molecule o f n u c l e i c  a c i d by s p l i t t i n g the s e n s i t i v e l i n k a g e s i n the i n t e r i o r of the c h a i n .  As such, they are d i s t i n g u i s h a b l e from  another  subgroup o f phosphodiesterases, the exonucleases which a t t a c k the c h a i n of n u c l e i c a c i d by a consecutive s p l i t t i n g of a mononucleotide  from one end o f the c h a i n .  DNases, as the name  i m p l i e s , a l s o show a q u a n t i t a t i v e p r e f e r e n c e toward  deoxyribo-  n u c l e o t i d e d e r i v a t i v e s , although t h i s s p e c i f i c i t y toward sugar moiety  i s not always a b s o l u t e  the  (1).  The d i s c o v e r y of enzymes capable o f degrading n u c l e i c a c i d s dates as f a r back as 1 9 0 3 . when A r a k i ( 2 ) observed i t was  p o s s i b l e to l i q u e f y g e l s o f thymus n u c l e i c a c i d w i t h  e x t r a c t s of l i v e r ,  spleen and thymus, but t h e i r u n e q u i v o c a l  e x i s t e n c e was not demonstrated first  that  until  c r y s t a l l i z e d ribonuclease.  1939>  when K u n i t z  (3)  Ten y e a r s l a t e r , the c r y s -  t a l l i z a t i o n of deoxyribonuclease from the pancreas was a c h i e ved, a l s o by K u n i t z ( 4 ) .  The pH optimum o f t h i s p a n c r e a t i c  DNase l i e s cles:e<. to 7 . but i t i s somewhat i n f l u e n c e d by the  -2-  nature o f the i o n s present i n the medium  (4).  The enzyme  a l s o r e q u i r e s d i v a l e n t c a t i o n s f o r a c t i v i t y ; the products o f the r e a c t i o n were shown by Sinsheimer  (5) and Laskowski  to terminate e x c l u s i v e l y i n 5'-phosphates.  Another  (6)  DNase  which has an optimum pH o f about 4.5 to 5*5 and i s i n h i b i t e d by the a d d i t i o n o f M g  ++  was observed i n spleen by Catcheside  and Holmes (7) and i n thymus by Maver and Greco ( 8 ) . These o b s e r v a t i o n s were soon confirmed and extended  to o t h e r t i s s u e s .  A n a l y s i s o f the products o f these two " a c i d " DNases by Koerner and Sinsheimer  (9) and Laskowski  (10) showed that the r e a c t i o n  products a l l c a r r y a phosphomonoester group a t the 3'-end. At present, the DNases are s u b d i v i d e d i n t o two c l a s s e s , the DNase I and DNase I I enzymes.  These two c l a s s e s were  i n t r o d u c e d by Cunningham and Laskowski  (11) to compare and  to c o n t r a s t enzymes d i s t r i b u t e d i n d i f f e r e n t t i s s u e s w i t h t h e i r p r o t o t y p e s w i t h r e s p e c t to pH optimum, behaviour w i t h a c t i v a t i n g and i n h i b i t o r y agents and the nature o f the products formed.  P a n c r e a t i c DNase o f K u n i t z (4) and the thymus DNase  o f Maver and Greco (8) served as p r o t o t y p e s f o r DNase I and DNase I I r e s p e c t i v e l y . purpose  T h i s terminology i s u s e f u l f o r the  o f c h a r a c t e r i z a t i o n o f enzymes i n d i f f e r e n t  I n accordance w i t h t h i s , the term  tissues.  " i n t e s t i n a l DNase I " i m p l i e s  an enzyme i s o l a t e d from the i n t e s t i n a l mucosa and resembling p a n c r e a t i c DNase i n p r o p e r t i e s .  -3B i o l o g i c a l F u n c t i o n o f Deoxyrlbonucleases Many t i s s u e s have been shown to possess an enzyme complement capable o f degrading n u c l e i c a c i d s  (11-16).  It  i s more than l i k e l y that the f u n c t i o n o f t h i s enzyme complement i s not a p a s s i v e  one, i . e . , i t does not represent  merely a means o f degrading n u c l e i c a c i d s i n dead o r l y s e d c e l l s so t h a t the products may be used as s u b s t r a t e s i n n u c l e i c a c i d s y n t h e s i s by l i v i n g members o f the c e l l population.  The p o s s i b i l i t y i s that i t performs a c t i v e l y i n the  i n t r a c e l l u l a r metabolism o f g e n e t i c a l l y and i n f o r m a t i o n a l l y active nucleic acids.  T h i s subject has r e c e i v e d  a t t e n t i o n i n recent y e a r s .  considerable  Although s t u d i e s i n t h i s  field  have been c o n f i n e d mainly to b a c t e r i a , from the g e n e r a l larities  simi-  i n b a s i c metabolic pathways, and the r e s u l t s o f  some s t u d i e s on higher organisms, i t may be i n f e r r e d that the r e s u l t s obtained higher  with microorganisms a r e a l s o a p p l i c a b l e to  organisms. In v i t r o  s t u d i e s c a r r i e d out by Romberg et a l . ( 1 7 ,  18) on the r e p l i c a t i o n o f DNA catalysed, by E s c h e r i c h i a  coli  DNA polymerases demonstrated that DNA s y n t h e s i s r e q u i r e s the presence o f 3'-hydroxyl groups i n the primer DNA. s y n t h e s i s o f the DNA-like primer dGdC by h i g h l y preparations  o f DNA polymerases occurs very  Thus,  purified  slowly,  i f at a l l ,  even when the a p p r o p r i a t e polymer s y n t h e s i s  However,  begins immediately upon the a d d i t i o n o f  very low c o n c e n t r a t i o n s by  primer i s p r o v i d e d .  o f endonuclease I .  Endonuclease I,  c l e a v i n g the DNA primer to produce a d d i t i o n a l  3*-hydroxyl  groups, g r e a t l y enhances i t s a b i l i t y to support r a p i d and ( 1 8 ) .  e x t e n s i v e DNA s y n t h e s i s  While i n d i s c r i m i n a t e  cleavage  o f phosphodiester bonds by DNA endonucleases i s i n i m i c a l to g e n e t i c  i n t e g r i t y , the s p e c i f i c i n t r o d u c t i o n o f 3 ' - h y d r o x y l  groups by a c a r e f u l l y c o n t r o l l e d e n d o n u c l e o l y t i c facilitate  DNA s y n t h e s i s  Circumstantial  f i s s i o n may  i n vivo.  evidence p o i n t i n g  to the involvement  o f DNases i n DNA r e p l i c a t i o n a l s o comes from c o r r e l a t i o n s t u d i e s : c e l l u l a r DNase l e v e l s a r e g e n e r a l l y , i n v a r i a b l y , highest when DNA s y n t h e s i s the  earliest  during  though not  t h a t i n t e r v a l i n the growth c y c l e  i s proceeding a t maximal r a t e .  Among  s t u d i e s r e l a t i n g l e v e l s o f DNase a c t i v i t y to  r a t e o f DNA s y n t h e s i s  i s one o f A l l f r e y and Mirsky  (13).  Upon comparing the DNase I I a c t i v i t i e s o f d i f f e r e n t organs o f the mouse, r a t , chicken,  c a l f and horse, these  investigators  observed a s i g n i f i c a n t c o r r e l a t i o n between the DNase content o f a p a r t i c u l a r t i s s u e and i t s c a p a c i t y or r e g e n e r a t i o n . the  Brody and T h o r e l l ( a < $ )  for proliferation a l s o observed i n  c h i c k e n a s i g n i f i c a n t l y h i g h e r l e v e l o f DNase I I a c t i v i t y  i n regenerating  bone marrow, as compared with normal.  There  was  a l s o a p o s i t i v e c o r r e l a t i o n i n these experiments  between m i t o t i c frequency and c e n t l y , Lehman et a l . (21, the v a r i o u s at v a r i o u s  E. c o l i  22)  enzyme a c t i v i t y .  examined the a c t i v i t i e s  of  DNases i n e x t r a c t s prepared from c e l l s  stages i n the growth c y c l e .  these enzymes were i n v a r i a b l y highest phase o f growth, and s t a t i o n a r y phase.  More r e -  The during  decreased to v a r y i n g  In the  activities the  of  exponential  degrees i n the  case o f endonuclease I,  enzymatic  a c t i v i t y a c t u a l l y dropped to undetectable l e v e l s when the c u l t u r e entered the  s t a t i o n a r y phase.  v a r i e t y of n u t r i t i o n a l conditions e r a t i o n time of the  Moreover, under a  which i n f l u e n c e d  c u l t u r e , there was  the gen-  a close r e l a t i o n s h i p  between the r a t e o f growth, r a t e o f DNA  synthesis  and  endo-  nuclease I a c t i v i t y . In a d d i t i o n to a p o s s i b l e t h e s i s , DNases may a protective  involvement i n DNA  a l s o serve the c e l l i n what may  capacity.  Thus Dussois and  that when c e l l s o f E. c o l i  s t r a i n K12  be  Arber (23)  termed  showed  were i n f e c t e d with  Xphage grown on a d i f f e r e n t host s t r a i n (B or C), a t r i c t i o n " p r o c e s s sets i n .  syn-  "res-  T h i s p r o c e s s b l o c k s phage mul-  t i p l i c a t i o n through r a p i d degradation o f the incoming DNA acid-soluble DNases.  fragments, presumably by the e x i s t i n g host  to  -6Another type o f p r o t e c t i v e mechanism a f f o r d e d by DNases i s the enzymatic r e p a i r o f damaged DNA. age may  Such dam-  r e s u l t from the i n t r o d u c t i o n i n t o the DNA  o f photo-  products, f o r example, p y r i m i d i n e dimers o r products formed by the a c t i o n o f v a r i o u s mutagens, i n c l u d i n g agents and X-rays (24, 2 5 ) .  alkylating  The r e p a i r p r o c e s s has been  s t u d i e d most thoroughly by Setlow and C a r r i e r  (26).  They  showed that the r e a c t i v a t i o n i s enzymatic i n nature and c o n s i s t s o f the e x c i s i o n , from the p o l y n u c l e o t i d e chain, o f n u c l e o t i d e s damaged as a r e s u l t o f u l t r a v i o l e t tion.  irradia-  They proposed t h a t , f o l l o w i n g e x c i s i o n o f the damaged  bases from one o f the two  strands o f DNA,  the n u c l e o t i d e s  complementary to those o f the i n t a c t o p p o s i t e strands are i n s e r t e d i n t o the gap so formed by the a c t i o n o f a DNA merase; the DNA  double h e l i x i s then r e s t o r e d by  poly-  joining  the l a s t phosphodiester l i n k . T h i s type o f model f o r dark r e a c t i v a t i o n o f u l t r a violet-damaged DNA  has l e d to the search f o r an e x c i s i o n  enzyme which i s a b l e to r e c o g n i z e p y r i m i d i n e dimers o r o t h e r damaged bases and e x c i s e them from the DNA oligonucleotide.  as p a r t o f an  Since c o n s i d e r a b l e d i s t o r t i o n o f the  double h e l i x and l o c a l i n t e r r u p t i o n o f normal base  DNA  pairing  i s expected to r e s u l t when, f o r example, a dimer i s formed  -7between adjacent recognize  pyrimidines,  the e x c i s i o n enzyme might  such d i s t o r t i o n s or loops r a t h e r than the damaged  base i t s e l f .  Endonucleases have already been i d e n t i f i e d i n  NeuEpspora c r a s s a by Lehman (27)  and  i n lamb b r a i n by Healy  et a l . ( 1 5 ) which are h i g h l y s p e c i f i c f o r polynucleotides.  single-stranded  I t i s noteworthy t h a t one  of the r a t  i n t e s t i n a l enzymes to be d e s c r i b e d l a t e r was a t t a c k heat-denatured DNA.  According  a l s o found to  to Lehman (27),  such  enzymes c o u l d serve as e x c i s i o n enzymes by v i r t u e o f t h e i r ability DNA  to seek out and  destroy unordered r e g i o n s w i t h i n  double h e l i x . I t has been p o i n t e d out by Howard-Flanders and  (28)  the  that the mechanism f o r the r e p a i r of u l t r a v i o l e t  d i a t e d DNA  may  irra-  be c l o s e l y r e l a t e d to t h a t by which recom-  b i n a n t s are formed i n g e n e t i c c r o s s e s . most probably  Boyce  Since  recombination  occurs by means of a breakage and  mechanism (29),  reunion  i t i s p o s s i b l e t h a t the i n i t i a l event i n  recombination c o n s i s t s o f the breakage o f a phosphodiester l i n k i n the DNA  double h e l i x .  T h i s cleavage may  be  by the same enzyme i n v o l v e d i n e x c i s i n g p y r i m i d i n e  catalysed dimers  and other damaged bases. S p e c i f i c i t y and Use  o f DNases as A n a l y t i c a l Reagents  N u c l e o l y t i c enzymes have o f t e n been s t u d i e d from  -8the p o i n t o f view o f u s i n g them as reagents f o r s t u d y i n g the s t r u c t u r e and n u c l e o t i d e sequence o f n u c l e i c a c i d s .  In  the case o f enzymes which a t t a c k RNA, Ochoa ( 3 0 ) has shown t h a t p u r i f i e d E. c o l i p o l y n u c l e o t i d e phosphorylase can rapi d l y phosphorolyse r a n d o m l y - c o i l e d p o l y r i b o n u c l e o t i d e s  such  as p o l y u r i d y l i c a c i d but ordered, hydrogen-bonded s t r u c t u r e s such as t-RNA are r e l a t i v e l y r e s i s t a n t to a t t a c k by t h i s enzyme.  T h i s p r o p e r t y o f p o l y n u c l e o t i d e phosphorylase  suggests t h a t the enzyme may be used i n s t u d y i n g the secondary s t r u c t u r e o f p o l y r i b o n u c l e o t i d e s . et a l . ( 3 1 )  More r e c e n t l y , H o l l e y  e l u c i d a t e d the primary s t r u c t u r e o f alanyl-t-RNA  by e x p l o i t i n g the h i g h degree o f s p e c i f i c i t y o f the two RNA depolymerases,  p a n c r e a t i c RNase and T^ RNase.  To i l l u s t r a t e the use o f enzymes a c t i v e towards DNA as an a n a l y t i c a l t o o l , the case o f E. c o l i exonuclease be c i t e d .  T h i s enzyme was found by Sinsheimer  may  ( 3 2 ) to be  s e l e c t i v e f o r s i n g l e - s t r a n d e d DNA and may thus be used f o r the i d e n t i f i c a t i o n o f t h i s conformation i n a DNA  sample.  The enzyme i s a l s o i n c a p a b l e o f a t t a c k i n g phosphodiester bonds between the l a s t two n u c l e o t i d e r e s i d u e s a t the 5 ' terminus o f a p o l y n u c l e o t i d e c h a i n ( 3 2 ) . w i t h i t s l a c k o f endonuclease  T h i s , coupled  a c t i v i t y , permits the use o f  the enzyme as an end-group reagent.  -9A major definitively polypeptide have  trend i n nucleic  establish product.  been a c h i e v e d  a colinearity Considerable  by Y a n o f s k y  recombination analysis  of  amino a c i d r e p l a c e m e n t s a n d by B r e n n e r e t peptides  al.  synthesized  conclusive  proof of  demonstration that acid  chain i s  E.  (34)  c o u l d be towards  et  (33)  mutants  through t h e i r  exactly  must  order of the  of  along  facilitated  internucleotide  The p r o b l e m o f DNase I a n d s p l e n i c authors.  T i t r a t i o n data of  Butler  (36)  mately  25%  on p a n c r e a t i c of a l l  the  polypeptide  enzymes  be h y d r o l y s e d . established through to  that  of  products vary  of  However, direct  nucleic  the  correschain. DNA  hydrolyse.  of  pancreatic  and o f  by  various  Little  indicated that  linkages  from m o n o n u c l e o t i d e s eight  and  approxi-  chromatographic  w i t h about  poly-  selectivity  internucleotide  The a p p l i c a t i o n o f  oligonucleotides  (35)  synthetase  attacking  has been a t t a c k e d  DNase I  available  the  order of  specificity  Kunitz  genetic  T4 p h a g e .  the  if  direction  contain  studies  bonds which they  linkage  DNase I I  which  to  its  in their  come from a  f o u n d w h i c h show a h i g h d e g r e e the  in this  codons a l o n g  same a s  is  gen& a n d  advances  by amber m u t a n t s  the  the  p r o t e i n tryptophan  colinearity  c o u l d be  of  al.  ooli  i n the  p o n d i n g amino a c i d r e s i d u e s Such a f e a t  a c i d r e s e a r c h today  can techniques (37)  nucleotide  -lore s i dues (38). The composition o f l a r g e and small was not u n i f o r m w i t h r e s p e c t bases. (39)  fragments  to frequency o f each o f the  The l a r g e r fragments were found by Overend and Webb  to be r i c h i n p u r i n e s ,  but i n small fragments,  d i n e s predominate (38). A systematic  pyrimi-  identification of  p r o d u c t s by Sinsheimer (37) e s t a b l i s h e d t h a t a l l f o u r o f the common mononucleotides were present and accounted f o r about 1% o f the t o t a l d i g e s t .  The most extensive  study o f the  d i n u c l e o t i d e p r o d u c t s i n the DNase I d i g e s t was that o f Sinsheimer (37, 40, 4 l ) who found f i f t e e n o f the s i x t e e n p o s s i b l e d i n u c l e o t i d e s c o n t a i n i n g major bases, d i n u c l e o t i d e was dpApC.  The m i s s i n g  He a l s o noted t h a t i n d i n u c l e o t i d e s  composed o f purine, and p y r i m i d i n e ,  the sequence d-pPypPu  predominated over the sequence d-pPupPy, and i n t e r p r e t e d t h i s to mean that the l a t t e r l i n k a g e i s p a r t i c u l a r l y s e n s i t i v e to p a n c r e a t i c  DNase I .  With r e g a r d  to s p l e n i c DNase I I , s i m i l a r product  a n a l y s e s have been c a r r i e d out by Koerner and Sinsheimer ( 9 ) . DNase I I was found to produce more mononucleotides, consider a b l y l e s s d i n u c l e o t i d e s and c o n s i d e r a b l y oligonucleotides.  more o f the h i g h e r  Concerning the i n f l u e n c e o f adjacent bases  on the s u s c e p t i b i l i t y o f i n t e r n u c l e o t i d e l i n k a g e s  to DNase I I  Koerner and Sinsheimer (9) a l s o a n a l y s e d the fragments from  -11DNase I I d i g e s t i n r e s p e c t to the t e r m i n a l s and t h a t DNase I I shows no p r e f e r e n c e p y r i m i d i n e bases adjacent  concluded  f o r s p e c i f i c purine  to the s e n s i t i v e  linkages.  an independent study, L a u r i l a and Laskowski (10) s e v e r a l d i n u c l e o t i d e s and one them f o r sequences.  The  and  trinucleotide  In  isolated  and  analysed  sequence d-PypPup has not been  found among the i d e n t i f i e d products  and i t was  concluded  t h a t t h i s l i n k a g e i s p r e f e r e n t i a l l y h y d r o l y s e d by DNase I I . S e v e r a l o t h e r enzymes capable  o f a t t a c k i n g DNA  in  an e n d o n u c l e o l y t i c manner have been i s o l a t e d from v a r i o u s sources and  t h e i r s p e c i f i c i t i e s studied.  Georgatsos and  Antonoglou (42) i s o l a t e d a DNase from Octopus v u l g a r ! s a preference  towards the d-pXpC bond.  A nuclease  from mung  bean has been p u r i f i e d and  c h a r a c t e r i z e d by  (43).  found to be most s e n s i t i v e  The  d-pApX bond was  the enzyme, f o l l o w e d by d-pTpX. obtained  Sung and  An endonuclease has  i n c r y s t a l l i n e form by A n f i n s e n from  aureus ( 4 4 ) .  I t was  Laskowski towards been  Staphylococcus  shown by Dekker and Dirksen  a t t a c k heat-denatured DNA  with  b e t t e r than n a t i v e DNA.  (45) to Laskowski  (1) reviewed the l i n k a g e s p e c i f i c i t y o f t h i s enzyme and r e p o r t e d t h a t bonds o f the type d-XpAp and d-XpTp were most susceptible. I n s p e c t i o n o f p e r t i n e n t l i t e r a t u r e such as quoted  -12a b o v e makes i t  clear  t h a t none o f  shows any marked d e g r e e o f l i n k a g e for as yet specific  u n d i s c o v e r e d enzymes  internucleotide  concern of n u c l e i c a c i d  mucosa o f  by L e e a n d Z b a r s k y later,  the  separable two  DNases e x a m i n e d so  specificity.  capable  of  far  The  search  degrading  DNA a t  bonds must r e m a i n a n imminent researchers.  The e x i s t e n c e o f intestinal  the  two d e o x y r i b o n u c l e a s e s  the  rat  (46).  in  the  has been demonstrated  In experiments  two d e o x y r i b o n u c l e a s e s  were  on D E A E - c e l l u l o s e columns.  t o be  previously  described  found to  be  physically  P u r i f i c a t i o n of  these  enzymes was a c h i e v e d by a c e t o n e powder e x t r a c t i o n ,  ion-  exchange chromatography on D E A E - c e l l u l o s e columns and p a r t i t i o n on h y d r o x y l a p a t i t e . the  intestinal  To d e t e r m i n e t h e  DNases,  thymus DNA w i t h t h e s e  the  of  the  to  enzymes u n d e r v a r i o u s  However,  c o u l d be drawn from t h e s e  Regulation of  the  no c l e a r c u t  analysed  at  the  ends  conclusions  Activities  great variety  trations of nucleolytic  frequencies  were  studies.  Deoxyribonuclease  Despite  then  of  calf  conditions  These were  c h a i n l e n g t h and base  oligonucleotides.  specificity  products of h y d r o l y s i s of  i s o l a t e d by c o l u m n c h r o m a t o g r a p h y . with respect  linkage  enzymes  a n d somtimes h i g h  in cells,  concen-  the n u c l e i c  acids  -13do not undergo any p r e c i p i t o u s breakdown except under extreme c o n d i t i o n s o f c e l l death and l y s i s .  Obviously,  mechanisms must e x i s t to keep t h i s powerful n u c l e o l y t i c potency under c o n t r o l .  Studies o f p a r t i a l l y p u r i f i e d en-  zymes have i n many i n s t a n c e s g i v e n c l u e s as t o how they may be immobilized,  m e t a b o l i c a l l y o r s p a t i a l l y , w i t h i n the c e l l .  For example, endonuclease I i n E. c o l i has been shown by Lehman e t a l . (22) to be q u a n t i t a t i v e l y i n h i b i t e d by very low  concentrations  o f RNA.  T h i s enzyme may thus be immo-  b i l i z e d i n v i v o , by combination with c e l l u l a r RNA.. Regulation  o f DNase a c t i v i t i e s a t the c e l l u l a r  l e v e l has a l s o been s t u d i e d by the i s o l a t i o n and i d e n t i f i cation of protein inhibitors  (47, 48) and by examining the  e f f e c t o f changes i n the i o n i c environment on enzyme a c t i vity. Mg  ++  I n 1946, and M n  ++  McCarty  (49) observed t h a t the presence o f  i n c r e a s e d the r a t e o f h y d r o l y s i s o f c a l f  thymus DNA by p a n c r e a t i c DNase I . L a t e r on, Kunitz (35) noted t h a t the requirement f o r M g  + +  i n c r e a s e s with i n c r e a s i n g  c o n c e n t r a t i o n o f s u b s t r a t e but i s independent o f the enzyme concentration. has be  He c o n s i d e r e d  i t p o s s i b l e t h a t the n u c l e a t e  to be i n the form o f a magnesium compound i n order t o s u s c e p t i b l e to DNase I . A s i m i l a r suggestion  was advanced  by Weissman and F i s h e r (50) on the b a s i s o f McCarty's data  -14( 4 9 ) , t h a t the c o n c e n t r a t i o n o f M g c l o s e r to t h a t o f the s u b s t r a t e the enzyme (2.5 x 10~^ M). 2  (3 x 10""-%) was  ++  (5 x 10~9M) than t h a t o f  The most thorough s t u d i e s o f  the interdependence o f i o n s and  substrate  are those o f Desreaux et a l . (51) and The  c o n c l u s i o n drawn from these  o f h y d r o l y s i s o f DNA  Shack and Bynum ( 5 2 ) .  s t u d i e s i s t h a t the r a t e  by p a n c r e a t i c DNase I depends on  c o n c e n t r a t i o n o f DNA, and  concentration  H+,  the  and mono- and d i v a l e n t c a t i o n s ,  t h a t a l l f o u r o f these parameters are  interdependent.  The a c t i v a t i o n o f p a n c r e a t i c DNase I by b i v a l e n t metals has been shown to be a very complex phenomenon. Erkama and DNA  and  by M g  ++  Suutarinen  (53) prepared  Mg  + +  and M n  t e s t e d them as s u b s t r a t e s f o r DNase I . a t 0.003M c o n c e n t r a t i o n was  s a l t s of  Activation  found to i n c r e a s e  r a t e o f h y d r o l y s i s o f MgDNA and MnDNA. prompted the authors  ++  the  These f i n d i n g s  (53) to conclude t h a t the mechanism of  the metal a c t i v a t i o n o f DNase I cannot be e x p l a i n e d  satis-  f a c t o r i l y by the concept o f m e t a l l o s u b s t r a t e alone,  as  suggested by Kunitz  (35).  Wibery (5^) observed that  Ca  + +  alone has but l i t t l e a c t i v a t i n g e f f e c t on p a n c r e a t i c DNase I . But when C a  + +  was  added to a system a l r e a d y c o n t a i n i n g M g ,  they produced a strong s y n e r g i s t i c e f f e c t . and Becking  ++  Work of  (55) showed t h a t the r e l a t i v e amounts o f  Hurst purines  -15-  and pyrimidines  occupying the 5'-terminal p o s i t i o n i n  fragments was dependent upon the nature of the ion present. Bollum (56) found that i n the presence of Mg  ++  alone, panc-  r e a t i c DNase I degrades only the poly-dC strand of the doublestranded homopolymer dI;dC. to a medium containing Mg  ++  However, the addition of C a resulted i n the digestion of  the d l as well as the dC strand. for the mixture of Mg  ++  + +  When Mn  ++  was substituted  and C a , the same r e s u l t was observed, ++  i . e . , both chains of the polymer were digested.  These expe-  riments suggested that the a c t i v i t i e s of DNases might be regulated i n vivo by changes i n the ionic environment. In experiments described i n t h i s thesis, i t was found that one of the two DNases (DNase I) of the i n t e s t i n a l mucosa of the rat showed an absolute requirement for bivalent metal ions for a c t i v i t y .  Q u a l i t a t i v e l y , i t was found that,  with t h i s enzyme, the kind of internucleotide linkages hydrolysed  depends upon the nature of the ions present.  Quan-  t i t a t i v e l y , optimal a c t i v a t i o n of the enzyme was shown to require a very delicate balance between metal and DNA concentrations. The mechanism of a c t i v a t i o n of the i n t e s t i n a l DNase I was  studied by k i n e t i c analysis of the reaction.  Several  authors (53, 57, 58) have shown that DNA binds bivalent  -16metal for  i o n s . Under s u c h  initial  f r e e metal cause the easily  velocities and  free  free  circumstances,  the r a t e  o f the  r e a c t i o n i n terms o f  DNA  c o n c e n t r a t i o n s cannot  concentrations o f these  found  liminary  t o be  too  experiments  metal  complicated  and  e q u a t i o n was  i n the p r e s e n t  developed  metallosubstrate,  derived  capable  The  rate was  Since  pre-  s t u d i e s showed t h a t  the t r u e s u b s t r a t e , a r a t e  i n terms o f the  first  o f combining metal  DNA  The  complex.  was  concentration of  simple  ions, free  second  and  DNA  equation  the  was  DNA  assumption  derivation of  intestinal and  and was  the  enzyme  simultaneously  a l s o w i t h the t h a t the  data  metal-  combination  W i t h i n a c e r t a i n range  c o n c e n t r a t i o n s , the k i n e t i c  equation well, On  t h a t the  independently  t a k e s p l a c e i n random o r d e r .  valid.  The  For the d e t e r m i n a t i o n o f the c o n c e n t r a t i o n  with bivalent  rate  system.  concentrations  a s s u m p t i o n s were made i n t h e  equation.  metal  not  from a c o n s i d e r a t i o n o f the d i s s o c i a t i o n e q u i l i b r i u m . Two  was  components a r e  analysed.  t h e metal-DNA complex, a r e l a t i v e l y  rate  be-  DNA  t o be  t h e metal-DNA complex i s p r o b a b l y  of  be u s e d  obtained i n a multiple equilibrium  e q u a t i o n i n terms o f t o t a l also  forward  equation  of  fitted  the  s u g g e s t i n g t h a t t h e a s s u m p t i o n s were  the b a s i s o f these k i n e t i c  Metal complex o f t h e t y p e Enzyme-' i was ^ DNA  studies, a ternary p o s t u l a t e d t o be  an  -17an i n t e r m e d i a t e  i n the i n t e s t i n a l DNase I r e a c t i o n .  The e f f e c t s o f EDTA, c i t r a t e and arsenate  on the  i n i t i a l v e l o c i t i e s o f the DNase I r e a c t i o n were a l s o s t u d i e d . By r e l a t i n g the extent o f i n h i b i t i o n to the c o n c e n t r a t i o n o f f r e e metal i o n s i n the presence o f v a r y i n g l e v e l s o f these  c h e l a t i n g agents, a mechanism f o r t h e i r  e f f e c t was p o s t u l a t e d .  inhibitory  I t was suggested t h a t c i t r a t e and  arsenate  i n h i b i t by removing the metal i o n s whereas EDTA  probably  e x e r t s i t s e f f e c t by forming  a metal-EDTA complex  which can compete with the metal-DNA complex f o r the enzyme.  -18I I . ISOLATION AND PURIFICATION OF TWO  DEOXYRIBONUCLEASES  FROM THE INTESTINAL MUCOSA OF THE RAT EXPERIMENTAL 1. Enzyme  Assays  DNase a c t i v i t i e s were assayed by the h y p e r c h r o m i c i t y method o f K u n i t z (4) which measures the i n c r e a s e i n absorbance o f a s o l u t i o n o f DNA a t 260 mu when i t i s s u b j e c t e d to treatment with DNase.  The measurements were made i n a  G i l f o r d 2000 Spectrophotometer  equipped with a constant-  temperature  Cuvettes c o n t a i n i n g 3 ml o f  c e l l compartment.  s u b s t r a t e s o l u t i o n were e q u i l i b r a t e d to 37C i n t h i s chamber. At zero time, 0.1 o r 0.2 ml o f enzyme s o l u t i o n were added r a p i d l y and mixed, and the change i n absorbance a c t i o n mixture monitored.  o f the r e -  One u n i t o f enzyme a c t i v i t y  was  d e f i n e d as the amount o f enzyme capable o f b r i n g i n g about an i n c r e a s e i n absorbance  o f 1.0 per min a t 260 mu under the  c o n d i t i o n s o f assay. The  s u b s t r a t e s o l u t i o n f o r assay o f i n t e s t i n a l  DNase I c o n t a i n e d c a l f thymus DNA a t a c o n c e n t r a t i o n o f 50 pg per ml i n 0.1M ammonium a c e t a t e b u f f e r , pH 6 . 8 , was a l s o 0.01M w i t h r e s p e c t to MgCl2«  which  When i t was l a t e r  found t h a t the DNase I r e a c t i o n was a c t i v a t e d to a g r e a t e r  -19extent by MnGl2, M n assay mixture.  ++  was s u b s t i t u t e d f o r M g  + +  i n the  For the assay o f i n t e s t i n a l DNase I I , the  s u b s t r a t e s o l u t i o n c o n t a i n e d 50 ug o f c a l f thymus DN'A p e r ml i n O.33M sodium formate b u f f e r , pH 4 . 5 . 2.  P r e p a r a t i o n o f Crude I n t e s t i n a l E x t r a c t F i v e o r ten male Wistar r a t s from the colony o f the  U n i v e r s i t y o f B r i t i s h Columbia, were s t a r v e d f o r 24 hours.  weighing 180 t o 2 0 0 g each,  Each was then k i l l e d by a blow  on the head and d e c a p i t a t e d .  The small i n t e s t i n e was removed  and c u t i n t o segments approximately 10 cm i n l e n g t h . segments were f l u s h e d f r e e o f contents w i t h c h i l l e d Ringer phosphate  These Krebs-  b u f f e r , pH 7 . 8 ( 5 9 ) . They were then  s p l i t open and a p p l i e d t o a c o l d g l a s s s u r f a c e w i t h the mucosal s i d e f a c i n g upwards.  The mucosal e p i t h e l i u m was  then scraped o f f with the edge o f a microscope  s l i d e and  p l a c e d i n a 100-ml graduated c y l i n d e r standing i n i c e . Enough Krebs-Ringer phosphate 1:10  b u f f e r was added to g i v e a  (v/v) suspension o f t i s s u e .  The suspension was homo-  g e n i z e d i n a S e r v a l l Omni Mixer a t 3000 r.p.m. f o r 15 mins a t OC.  The homogenate was c e n t r i f u g e d a t 1 0 5 , 0 0 0 x g f o r  60 mins a t 0C i n the Spinco Model L u l t r a c e n t r i f u g e .  The  supernatant s o l u t i o n c o n s t i t u t e d the crude enzyme e x t r a c t .  -203. P r e p a r a t i o n of Acetone Powder In e a r l y p u r i f i c a t i o n experiments, the crude enzyme e x t r a c t was  f r a c t i o n a t e d by a p p l y i n g i t d i r e c t l y to i o n -  exchange columns.  L a t e r on,  i t was  found b e n e f i c i a l to pre-  pare an acetone powder from the crude enzyme e x t r a c t p r i o r to chromatography.  T h i s g i v e s a c l e a n e r and more  concentrated  enzyme p r e p a r a t i o n . A l l the procedures were c a r r i e d out at 4C  according  to a method d e s c r i b e d by Georgatsos and Antonoglou Acetone was  p r e c h i l l e d a t -20C.  e x t r a c t prepared c h i l l e d acetone. -20C  above was  One  volume o f the crude  s t i r r e d slowly i n t o 40 volumes o f  The p r e c i p i t a t e was  allowed  f o r 30 mins, a f t e r which the supernatant  discarded.  (42).  The acetone powder was  to s e t t l e at liquid  was  recovered by c e n t r i f u g a t i o n  4n the S e r v a l l BC2B r e f r i g e r a t e d c e n t r i f u g e f o r 20 mins a t 4000 x g.  The p r e c i p i t a t e was  and a g a i n recovered  washed with 5°° ml of acetone  by c e n t r i f u g a t i o n .  peated once more, and  The washing was  the p r e c i p i t a t e was  acetone by l y o p h i l i z a t i o n .  The  re-  r i d a f excess  dry, pink acetone powder  was  e x t r a c t e d by s t i r r i n g i t with a minimum volume o f Cf.olM Tris(hydroxymethyl)aminomethane h y d r o c h l o r i d e b u f f e r , pH to b r i n g i t i n t o s o l u t i o n .  A f t e r 2 hours a t 4C,  amount of u n d i s s o l v e d m a t e r i a l was and  discarded.  the  7.8,  small  removed by c e n t r i f u g a t i o n  -214.  D e s a l t i n g with Sephadex G-25  Columns  D e s a l t i n g o f p r o t e i n s o l u t i o n s was a c h i e v e d by g e l (60).  filtration Sweden) was  Sephadex G-25  suspended  powder (Pharmacia, Uppsala,  i n d i s t i l l e d water to s w e l l .  then washed s e v e r a l times to remove f i n e s .  A slurry  p r e p a r e d from 10 v o l s o f water and 1 v o l o f g e l . (1 x 20 cm) was  was  The column  then packed a t atmospheric p r e s s u r e .  sample ( l e s s than 4 ml) to be d e s a l t e d was the  I t was  The  then poured onto  column and the p r o t e i n e l u t e d with d i s t i l l e d water.  The  f r a c t i o n s were c o l l e c t e d , assayed f o r u l t r a v i o l e t - a b s o r b i n g m a t e r i a l a t 280 mu and processed as d e s c r i b e d 5.  later.  Ion Exchange Chromatography Batches o f D E A E - c e l l u l o s e purchased from three d i f f e -  r e n t manufacturers have been used. B e r l i n , N.H.  M a t e r i a l from Brown Co.,  and from BioRad L a b o r a t o r i e s ( C e l l e x D, 0 . 9 5  per g) were prepared f o r chromatography  as f o l l o w s .  meq  The  exchanger was washed r e p e a t e d l y w i t h water to get r i d o f fines.  I t was  b u f f e r , pH 7.8, of  1.0N.  then suspended  i n a s o l u t i o n o f 0.01M  Tris  which a l s o c o n t a i n e d NaCl a t a c o n c e n t r a t i o n  A column was packed to a dimension o f 4x40cm from  t h i s suspension under a p r e s s u r e o f 5 p . s . i .  The DEAE-cellu-  l o s e was  then washed i n the column w i t h 4M. NaCl i n T r i s  buffer.  An a d d i t i o n a l 4 . 5  l i t e r s o f T r i s b u f f e r was  run  -22through the column to remove excess C l ~ . In e a r l i e r experiments, operated a t 4C. to  2x90  the column was packed and  I n l a t e r runs, the column s i z e was changed  cm to e f f e c t b e t t e r  resolution.  equipped w i t h an o u t e r temperature  These columns were  jacket,  and through the  e n t i r e p r o c e s s o f packing and e q u i l i b r a t i n g ,  the column was  kept c o o l by running tap water through t h i s  jacket.  When D E A E - c e l l u l o s e from Whatman Co. (DE22, form) was used,  the d i r e c t i o n s  o f the manufacturer  f o l l o w e d i n the p r e p a r a t i o n o f the exchanger.  fibrous were  Two hundred  and f i f t y g o f the c e l l u l o s e were s t i r r e d i n 4 l i t e r s o f 0.5N  HC1 and l e f t  standing f o r 30 to 60 mins.  The supernatant  l i q u o r was decanted o f f and the suspension washed with water i n a large  f u n n e l u n t i l the pH o f the f i l t r a t e  The c e l l u l o s e was then t r e a t e d and l e f t  standing f o r a f u r t h e r  w i t h 4 l i t e r s o f 0.5N NaOH 30 mins.  s o l u t i o n was d i s c a r d e d and a f u r t h e r 0.5N  reached 4 . 0 .  NaOH was added to the c e l l u l o s e .  The supernatant  volume o f 4 l i t e r s o f A f t e r another 30 mins,  the supernatant l i q u i d was a g a i n removed and the c e l l u l o s e was  washed r e p e a t e d l y w i t h water u n t i l the e f f l u e n t  showed  a constant pH w i t h washing.  The c e l l u l o s e was then s t i r r e d i n t o t i o n o f NaCl to b r i n g  sufficient  solu-  the f i n a l volume o f the s l u r r y t o 4  -23liters, The  the c o n c e n t r a t i o n o f NaCl i n the s l u r r y being 0.0625M.  s l u r r y was then t i t r a t e d slowly with constant  to pH 7.8 w i t h 0.0625N HCI. decanted o f f a f t e r one hour.  stirring  The supernatant s o l u t i o n was The r e s i d u e was washed twice  w i t h 4 l i t e r s o f O.O625M T r i s b u f f e r , pH 7.8.  Each time,  the c e l l u l o s e was a l l o w e d to stand i n the b u f f e r f o r one hour b e f o r e removing exchanger  the supernatant s o l u t i o n .  The e q u i l i b r a t e d  was then s t i r r e d i n t o 18 l i t e r s o f 0.61M  b u f f e r , pH 7.8 and a l l o w e d to s e t t l e f o r 30 mins.  Tris The super-  natant l i q u i d was poured o f f and the p r o c e s s repeated. exchanger  The  was then poured i n t o 0.01M T r i s b u f f e r , pH 7.8 to  make an approximately $% suspension (w:v).  A s l i g h t vacuum  was a p p l i e d to the c o n t a i n e r to remove o c c l u d e d a i r . The column was packed to dimensions o f 2x90 cm from t h i s s l u r r y under a p r e s s u r e o f 5 p.s.fc. as b e f o r e .  Column  s e t t i n g was e f f e c t e d by p a s s i n g through 0.01M T r i s b u f f e r , pH 7.8. The enzyme s o l u t i o n was a p p l i e d to the column, and a continuous flow o f 0.01M T r i s b u f f e r , pH 7.8, was begun a t a r a t e o f about 1 ml per min. and the absorbance  F r a c t i o n s o f 12 ml were c o l l e c t e d  o f each f r a c t i o n was measured a t 280 mp.  P r o t e i n s which were c a t i o n i c under these c o n d i t i o n s emerged immediately a f t e r displacement o f the holdup volume.  When  -24the c a t i o n i c p r o t e i n s had been c o l l e c t e d and the absorbance a t 280 was  mu  had r e t u r n e d to a b a s e - l i n e value, the washing 1 l i t e r of b u f f e r f o r  continued with another  discussed i n Results.  A l i n e a r g r a d i e n t from 0 to  NaCl i n Q:.01M T r i s b u f f e r , pH 7.8 was elution.  F r a c t i o n s of 12  0.5M  then e s t a b l i s h e d f o r  ml each were c o l l e c t e d i n a r e -  frigerated fraction collector. assayed  reasons  f o r DNase a c t i v i t y .  Representative  f r a c t i o n s were  A c t i v e f r a c t i o n s were pooled,  concentrated by l y o p h i l i z a t i o n and d e s a l t e d by g e l f i l t r a t i o n on "a' .columnlo f Sephadex G - 2 5 .  The  r  e f f l u e n t was  made up  0.002M with r e s p e c t to phosphate and a d j u s t e d to pH 6. H y d r o x y l a p a t i t e  to  6.8.  Chromatography  H y d r o x y l a p a t i t e was t o r i e s , Richmond, C a l i f .  purchased from BioRad  ( B i o g e l HTP).  I t was  Labora-  made i n t o a  t h i n s l u r r y with 0.001M phosphate b u f f e r , pH 6.8.  A column,  1x10  passing  cm,  was  packed from t h i s s l u r r y and washed by  through 25 ml of 0.001M phosphate b u f f e r . to stand o v e r n i g h t a t The  then  enzyme s o l u t i o n o b t a i n e d from Step 5 was  applied  A f t e r a d s o r p t i o n of p r o t e i n ,  c a r r i e d out i n a stepwise  b u f f e r s (pH 6.8)  allowed  4C.  to the h y d r o x y l a p a t i t e column. e l u t i o n was  I t was  manner with phosphate  of the f o l l o w i n g c o n c e n t r a t i o n s : 0 . 0 0 5 M ,  -250.01M, 0.03M, 0.05M, 01.075M, O.IM, p r e s s u r e o f 1.7  to 1.8  p . s . i . was  0.2M,  0.3M  A  a p p l i e d during e l u t i o n  f r a c t i o n s o f ten ml each were c o l l e c t e d . assayed  and 0.4M.  Each f r a c t i o n  and was  f o r p r o t e i n content by the method of Lowry et a l . ( 6 l )  and f o r DNase a c t i v i t i e s . 7.  L i g a t i n g of P a n c r e a t i c Duct of Rat C e r t a i n experiments (see l a t e r ) w i t h both the crude  i n t e s t i n a l e x t r a c t and p u r i f i e d p r e p a r a t i o n s showed that  one  of the i n t e s t i n a l DNases resembled p a n c r e a t i c DNase I to a considerable extent.  The p o s s i b i l i t y was  r a i s e d that  this  enzyme might have o r i g i n a t e d from p a n c r e a t i c j u i c e which been shown by K e l l e r et a l . (62) to c o n t a i n a h i g h r a t i o n o f p a n c r e a t i c DNase I .  To e l i m i n a t e t h i s  were used. with ether.  During the o p e r a t i o n , the animal was The abdomen o f the r a t was  performed.  200  to 225  S  anaethesized  shaved with a r a z o r  blade and rubbed with 1:1000 s o l u t i o n of z e p h i r a n . was  concent-  possibility,  the l i g a t i n g of the p a n c r e a t i c duct of the r a t was Male Wistar r a t s weighing approximately  has  The  skin  s l i t open f o r a l e n g t h of about \ i n c h along the median  l i n e o f the abdomen.  A t i n y hole was  then cut through  muscle l a y e r underneath and the h o l e was  carefully  the  enlarged  w i t h a b l u n t p a i r of s c i s s o r s , t a k i n g care not to damage the liver.  The  hole was  h e l d open with a r e t r a c t o r and  the  -26duodenum l o c a t e d .  The common p a n c r e a t i c - b i l e duct was  u s u a l l y found about 2 cm d i s t a l to the p y l o r i c s p h i n c t o r and embedded i n p a n c r e a t i c t i s s u e .  The duct was c a r e f u l l y  freed  from the surrounding connective and p a n c r e a t i c t i s s u e s a t the p o i n t where i t e n t e r s the duodenum.  A l i g a t u r e was made  w i t h s i l k t h r e a d around the duct a t t h i s p o i n t and the i n t e s t i n a l l o o p s r e p l a c e d i n the abdominal  cavity.  The r e t r a c t o r  was  then withdrawn and the muscle  l a y e r s sewn up.  was  then sutured and the wound cleaned w i t h z e p h i r a n s o l u t i o n .  The whole o p e r a t i o n r e q u i r e d about 45 mins.  The s k i n  The r a t s were  allowed to starve f o r 24 hours and were k i l l e d . RESULTS AND DISCUSSION F i g . 1 shows the chromatographic r e s o l u t i o n o f the a n i o n i c p r o t e i n s o f a crude i n t e s t i n a l mucosal DEAE-cellulose.  e x t r a c t on  I t can be seen t h a t two peaks o f deoxyribo-  n u c l e a s e s a c t i v i t i e s were found i n the e f f l u e n t f r a c t i o n s .  One  enzyme was e l u t e d very e a r l y i n the a n i o n i c f r a c t i o n , the o t h e r a t about 0.15M NaCl.  The former enzyme was found to  have an optimum pH o f 6 . 5 to 6 . 8 and to r e q u i r e b i v a l e n t metals f o r a c t i v i t y .  Thus i t f a l l s i n t o the category o f  enzymes termed DNase I by Cunningham and Laskowski  (11).  The  second enzyme was found to have an a c i d i c optimum pH ( 3 . 5 to 4.0). . I t does not r e q u i r e b i v a l e n t metal i o n s f o r a c t i v i t y .  0  20  40  60  80  100  120  140  160  180  T U B E oNO. F i g . 1. Chromatography f crude i12 n t eml. s t i n a l mucosal e x t r a c t on D E A E - c e l l u l o s e , DNase a c t i v i t y i s e x p r e s s e d i n A.o,D.. p e r min ( x l O ) and i s i n d i c a t e d by t h e shaded areas. Twelve-rnl f r a c t i o n s were c o l l e c t e d v - 2  :  Fig.  TUBE  NO.  2. Chromatography o f acetone powder e x t r a c t o f i n t e s t i n a l DNases on D E A E - c e l l u l o s e . Shaded a r e a s i n d i c a t e DNase activity. F i v e ~ m l f r a c t i o n s were c o l l e c t e d .  -29-  I t therefore belongs to the class of enzymes termed DNase II  (11).  F i g . 2 shows the elution pattern of ion exchange chromatography  on DEAE-cellulose of an acetone powder extr-  act of the i n t e s t i n a l enzymes.  I t i s seen that immediately  a f t e r displacement of the hold-up volume, a large cationic protein peak was washed o f f .  Further washing with buffer  eluted two small peaks of protein, the f i r s t of which  was  yellow i n color and was quite e a s i l y seen as i t moved down the column.  The second peak contained DNase I a c t i v i t y .  The active fractions have a low protein content.  Thus by  not starting the elution immediately a f t e r the main cationic peak was washed , the s p e c i f i c a c t i v i t y of the DNase I preparation was improved. 0.15M  DNase II was again eluted at about  NaCl. In experiments where the crude extract was chroma-  tographed d i r e c t l y , i t was found necessary to subject the active eluant fractions to further p u r i f i c a t i o n on hydroxylapatite columns.  Figures 3 and 4 present the elution diagrams  of DNase I and DNase II respectively from such columns.  The  i n t e s t i n a l DNase I was found to be eluted between 0 . 0 5 and O.O75M phosphate concentration. phosphate.  DNase II was eluted at 0.3M  On storage of the enzyme at -20C, the phosphate  M  Phosphate  0.005 0.01  0-03 0.05 0.075| 0.1 [ 0.2 | 0.3 | 0.4  effluent Fig.  3.  Chromatography o f r a t i n t e s t i n a l DNase I on h y d r o x y l a p a t i t e column. Shaded a r e a i n d i c a t e s DNase a c t i v i t y . Numbers on t o p o f diagram i n d i c a t e m o l a r i t y o f phosphate b u f f e r s u s e d i n e l u t i o n .  0.15  phosphate 0.005 0.01 |0.03|0.O5 |0.075  c E Q  0.10  6 <  CD CL  £  0  o <  4  o  0-05 $  CL  cc  6)02 E  0  50 F i g . 4.  1  100 ml.  150  200  effluent  Chromatography o f r a t i n t e s t i n a l DNase I I on hydroxy 1a p a t i t e column. Shaded, a r e a shows DNase I I a c t i v i t y . Numbers on t o p o f diagram i n d i c a t e m o l a r i t y o f phosphate i n e l u t i n g b u f f e r s .  i  -32was found to c r y s t a l l i z e out from s o l u t i o n centration  a t t h i s con-  (0.3M) but t h i s d i d not seem a f f e c t  v i t y o f DNase I I .  The r e s u l t s o f a t y p i c a l  the a c t i -  purification  experiment are summarized i n Table I . Table I .  P u r i f i c a t i o n of I n t e s t i n a l  Fraction  DNases.  Total units of activity  Total protein (mg)  Specific activity ( u n i t s per mg p r o t e i n )  Crude e x t r a c t  26.5  988  0.027  DEAE-cellulose + Sephadex G-25  37.2  7.13  5.2  Hydroxylapatite  33.8  0.335  101  Crude e x t r a c t  25.3  988  0.025  DEAE-cellulose + Sephadex G-25  21.5  19  1.12  Hydroxylapatite  17.0  1.28  13.3  DNase I  DNase I I  I t can be seen from Table I that the i n t e s t i n a l DNase I was p u r i f i e d about 3 , 0 0 0 f o l d , DNase I I about 500 fold.  I t i s also interesting  to note that the y i e l d o f  DNase I from the p u r i f i c a t i o n procedures i s about 1 2 0 $ ,  -33suggesting t h a t some enzyme i n h i b i t o r s might have been removed d u r i n g chromatography.  Although such i n h i b i t o r s  have not been demonstrated i n the r a t i n t e s t i n e ,  several  o t h e r t i s s u e s , e.g. r a t serum, c a l f spleen have been shown to c o n t a i n p r o t e i n i n h i b i t o r s o f DNase I ( 6 3 , 64) and DNase I I , (65,  66).  These i n h i b i t o r s may p l a y important r o l e s i n  r e g u l a t i n g c e l l u l a r DNase a c t i v i t i e s . I n e a r l y p u r i f i c a t i o n procedures, D E A E - c e l l u l o s e (Brown Co., B e r l i n ) was used w i t h success.  In l a t e r  experi-  ments, C e l l e x D from BioRad Lab. was used.  However, w i t h  t h i s r e s i n , i t was found t h a t the DNase a c t i v i t i e s a p p l i e d onto the column c o u l d not be r e c o v e r e d from the e f f l u e n t . No e x p l a n a t i o n can be o f f e r e d although i t i s q u i t e p o s s i b l e that i n h i b i t o r s o f the type d e s c r i b e d above were not separated from the DNases by C e l l e x D, thus r e s u l t i n g i n the l o s s o f a c t i v i t i e s o f the l a t t e r .  T h i s p o s s i b i l i t y was not pursued  f u r t h e r s i n c e the use o f Whatman DE 22 i o n exchanger i n f u r t h e r p u r i f i c a t i o n experiments proved  satisfactory.  To e s t a b l i s h the i n t e s t i n a l o r i g i n o f the DNases i s o l a t e d , the p a n c r e a t i c ducts o f the r a t s were l i g a t e d and the mucosal e x t r a c t prepared from these r a t s 24 hours a f t e r surgery.  The DNase a c t i v i t y o f such an e x t r a c t d i d not  d i f f e r s i g n i f i c a n t l y from that o b t a i n e d w i t h normal  rats,  i n d i c a t i n g t h a t the enzymes were o r i g i n a l l y p r e s e n t i n the  -34mucosal t i s s u e . that  the  T h i s was  nuclease  were a s s o c i a t e d  activities  w i t h the  The p a n c r e a s source  of  DNase I .  Dnase I I  has  Thus,  have  of  vely  secreting  (66).  (probably tissues levels  DNase I )  (pancreas, of  DNase I  digestive  enzyme.  glands  type  DNase I I ,  types of  glands.  on the  a c i d DNase a c t i v i t i e s  calf,  mouse a n d r a t  capacity  other  to  find  that  a digestive  proliferative that  it  contains  of  It  acti-  activity secretory  contain this  high  may be  of  d i f f e r e n t organs o f  The  high levels  therefore of  not  the  corre-  of a p a r t i c u l a r tissue  is  a  (13)  A l l f r e y and Mirsky  and  its  intestinal  f u n c t i o n and i n a d d i t i o n ,  tissue.  (14)  them  and observed a s i g n i f i c a n t  DNase c o n t e n t  and  h a n d , may be more  f o r p r o l i f e r a t i o n and r e g e n e r a t i o n .  mucosa s e r v e s highly  the  extracts  to  been  Shack  In general,  suggesting  compared t h e  l a t i o n between  con-  enzyme.  DNase  appear  concerned with c e l l u l a r metabolism.  horse,  shown t o  (68).  aqueous  glands)  enzyme,  best  B o t h DNase I  had h i g h e r  than r e s t i n g salivary  the  s t r a i n A mouse a n d f o u n d  found that  salivary  cell.  two u r i n a r y DNases have  c o n t a i n b o t h DNase I a n d DNase I I  (69)  showed  mucosa  i n the  been  p e p p r t e d i n human p l a s m a  Chepinoga  who  intestinal  matters  tissues  by p a p e r e l e c t r o p h o r e s i s were a l s o  rat  (67)  l o n g been r e c o g n i z e d as  DNases.  e x a m i n e d numerous t i s s u e s to  i n the  particulate  But most  t a i n both types of separated  c o n f i r m e d by R o b e r t s  it  is  a  surprising  both types of  DNases.  -35-  III.  SPECIFICITY OF THE RAT  INTESTINAL DEOXYRIBONUCLEASES  To i n v e s t i g a t e the p o s s i b i l i t y that the two r a t i n t e s t i n a l DNases might prove u s e f u l f o r the e l u c i d a t i o n of the s t r u c t u r e and n u c l e o t i d e sequence of DNA, s p e c i f i c i t y of the enzymes was  examined.  the l i n k a g e  These s t u d i e s  i n v o l v e d the i s o l a t i o n and i d e n t i f i c a t i o n of the products of the DNase r e a c t i o n .  The experimental procedures and  results  o b t a i n e d are d e s c r i b e d here. MATERIALS H i g h l y polymerized c a l f thymus DNA,  E. c o l l phos-  phomonoesterase and c a l f spleen phosphodiesterase were purchased from Worthington Biochem. Corp., Freehold, N.J. venom phosphodiesterase was  Snake  prepared from l y o p h i l i z e d C r o t a l u s  adamenteus venom (Ross A l l e n R e p t i l e I n s t . , S i l v e r Springs, F l o r i d a ) by the method of Koerner and Sinsheimer f u r t h e r p u r i f i e d a c c o r d i n g to F e l i x et a l . ( 7 0 ) . G-25  was a product of Pharmacia,  Uppsala, Sweden.  (9) and Sephadex DEAE-  c e l l u l o s e was purchased from BioRad L a b o r a t o r i e s , Richmond, C a l i f , under the brand name of c e l l e x - D . EXPERIMENTAL 1.  P r e p a r a t i o n of DNA  Hydrolysate  For the deoxyrlbonuclease I r e a c t i o n , 200 mg of c a l f  -36thymus DNA were d i s s o l v e d i n 200 ml o f 0.1M ammonium acetate  b u f f e r , pH 6.8,  which was a l s o 0.01M i n MgCl o r  MnCl2.  F i v e ml o f the p a r t i a l l y p u r i f i e d i n t e s t i n a l DNase I  2  s o l u t i o n were then added and the mixture i n c u b a t e d a t 3 7 C To m a i n t a i n o p t i m a l c o n d i t i o n s necessary to a d j u s t NaOH. ase  f o r enzyme a c t i v i t y , i t was  the pH p e r i o d i c a l l y by a d d i t i o n o f 0.1N  The r e a c t i o n was a l s o f o l l o w e d  by observing  i n absorbance o f the mixture a t 260 mu.  After  the i n c r e incubation  f o r approximately 7 hours, the r e a c t i o n mixture was heated a t 100C  f o r 10 min, cooled and f i l t e r e d .  The f i l t r a t e c o n s t i -  t u t e d the DNase I d i g e s t . When heat-denatured DNA was used as the s u b s t r a t e , a volume o f a s o l u t i o n o f heat-denatured DNA (see l a t e r mixed with an equal volume o f 0.2M ammonium a c e t a t e containing  0.02 moles MgCl2 per l i t e r .  ) was  buffer  The mixture was ad-  j u s t e d to pH 6.8 i f necessary and t r e a t e d with the i n t e s t i n a l enzyme as d e s c r i b e d  above.  When i n t e s t i n a l DNase I I was used, the s u b s t r a t e s o l u t i o n contained  1 mg o f DNA i n 1 ml o f 0.33M sodium  formate b u f f e r , pH 4 . 5 . 2>.  Ion-exchange Chromatography o f DNA The  hydrolysate  o l i g o n u c l e o t i d e s which r e s u l t e d from the above  -37digestion  were separated by ion-exchange chromatography  a c c o r d i n g to Tomlinson and Tener ( 7 1 ) .  B r i e f l y , a column  (2 x 90 cm) o f D E A E - c e l l u l o s e i n the c h l o r i d e  form was packed  under a p r e s s u r e o f 5 p . s . i . and e q u i l i b r a t e d w i t h 7M urea c o n t a i n i n g 30 ml o f 0.1M T r i s - H C l , loading,  pH 7 . 8 , p e r l i t e r .  After  e l u t i o n was a c h i e v e d by a l i n e a r g r a d i e n t o f NaCl  formed by two l i t e r s o f b u f f e r e d 7M urea i n the mixing chamber and 2 l i t e r s o f b u f f e r e d 7M urea, 0.5M with r e s p e c t to NaCl, i n the r e s e r v o i r . When M g  ++  was used i n the d i g e s t i o n  o f DNA, the  h y d r o l y s a t e a f t e r removal o f p r o t e i n by h e a t i n g c o u l d be loaded d i r e c t l y onto the column.  However, when M n  was the  ++  a c t i v a t i n g i o n , i t was found that a t the a l k a l i n e pH used f o r e l u t i o n , a brown g e l o r powder was formed on the r e s i n as w e l l as i n the e f f l u e n t .  T h i s brown p r e c i p i t a t e i s probably  Mn(0H)2 o r Mn02 and i t s presence i n t e r f e r e d s e r i o u s l y the  e l u t i o n and spectrophotometric a n a l y s i s  cleotides.  with  o f the o l i g o n u -  D i l u t i o n o f the h y d r o l y s a t e with water to reduce  the  concentration of Mn  the  f o r m a t i o n o f the p r e c i p i t a t e .  ++  p r i o r to l o a d i n g  d i d not a b o l i s h  S e v e r a l methods o f d e s a l t i n g  were attempted without much success.  For example,  t r a t i o n on Sephadex G - 2 5 columns d i d not g i v e c l e a n  gel-filseparation;  i t was a l s o accompanied by a l o s s o f mononucleotides.  When  an attempt was made to p r e c i p i t a t e the Mn++ from the DNA  -38-  hydrolysate by adding NH^OH, the oligonucleotides were prec i p i t a t e d simultaneously with Mn(0H)2.  Adsorption of the  oligonucleotides onto charcoal followed by elution with a l c o h o l i c ammonia was also found to be unsuccessful. i t was found that the p r e c i p i t a t i o n of Mn  ++  Finally,  on the column  could be prevented i f a chelating agent such as EDTA was also present i n the solution.  Therefore, p r i o r to loading onto  the column, the DNA hydrolysate was charged with EDTA to a f i n a l concentration of 0.05M.  After loading, the Mn-EDTA  complex was washed o f f the column with buffered 7M urea solution before e l u t i o n . A t y p i c a l elution pattern for the separation of oligonucleotides i s shown i n F i g . 5 . solved peaks were usually obtained.  Four f a i r l y well reHowever, to effect  clean separation of the dinucleotide f r a c t i o n (Peak II i n Fig.  5) from the t r i n u c l e o t i d e s (Peak I I I ) , i t was necessary,  a f t e r removal of urea, to rechromatograph the oligonucleotides at the overlapping region on small columns. 3.  Removal of Urea ( 7 1 ) The f r a c t i o n s comprising a peak were pooled and  d i l u t e d with 5 volumes of d i s t i l l e d water and adjusted to pH 8 i f necessary. DEAE-cellulose  The solution was then passed through a  (carbonate form) column the size of which  -39-  250  300  T U B E NO.  Chromatography o f a DNA h y d r o l y s a t e on DEAEcellulose. Mn was the a c t i v a t i n g i o n i n reaction, Peak I : m o n o n u c l e o t i d e s ; Peak I I : d i n u c l e o t i d e s ; Peak I I I : t r i n u c l e o t i d e s ; Peak I V : o l i g o n u c l e o t i d e s . + +  350  -40depended upon the amount o f o l i g o n u c l e o t i d e s loaded.  For  example, f o r 500 absorbance u n i t s * o f o l i g o n u c l e o t i d e s , a column 2x30 cm was used.  A f t e r l o a d i n g , the urea was washed  o f f with water; c h l o r i d e i o n s were removed with 0.02M ammonium carbonate.  The n u c l e o t i d e s were e l u t e d with 2M ammonium  carbonate, and the n u c l e o t i d e e f f l u e n t was f o l l o w e d by spectrophotometric measurements a t 260 mu.  The  UV-absorbing  f r a c t i o n s were pooled and evaporated to dryness i n a f l a s h evaporator a t a bath temperature  below 30C.  Water was added  to the r e s i d u e and the s o l u t i o n a g a i n concentrated to dryness. The process was repeated u n t i l no ammonium carbonate  remained.  The r e s i d u e was taken up i n a volume o f water (5 to 50 ml) depending upon the amount o f n u c l e o t i d e m a t e r i a l i n the f r a c tion. 4. Determination o f Chain Length o f O l i g o n u c l e o t i d e s T h i s was done by e s t i m a t i n g the r a t i o o f t o t a l phosphorus to phosphomonoesterase-sensitive o l i g o n u c l e o t i d e sample.  phosphorus i n the  A volume erf o l i g o n u c l e o t i d e s o l u t i o n ,  estimated to c o n t a i n about 2 - 3 ^ig o f m o n o e s t e r i f l e d phosphorus was mixed with an equal volume o f 0".'02M T r i s b u f f e r i  * One absorbance u n i t o f o l i g o n u c l e o t i d e s i s d e f i n e d as the amount o f m a t e r i a l which g i v e s r i s e to an absorbance o f one a t 260 mu when present i n 1 ml o f s o l u t i o n and when measured i n a cuvette w i t h l i g h t path = 1 cm.  -41s o l u t i o n , pH 8.0, which a l s o c o n t a i n e d MgCl2 a t a concent r a t i o n o f 0.02M.  To t h i s mixture, 0.01 ml o f an E. c o l i  phosphomonoesterase suspension was added, and the s o l u t i o n i n c u b a t e d a t 37C f o r 16 hours.  The r e a c t i o n was then stopped  by b o i l i n g the s o l u t i o n f o r 10 mins. temperature,  A f t e r c o o l i n g to room  the mixture was a n a l y s e d f o r l i b e r a t e d phos-  phate by the method o f F i s k e and SubbaRow ( 8 6 ) . T o t a l phosphate  was estimated i n a s i m i l a r way ( 8 6 )  a f t e r d i g e s t i o n o f the o l i g o n u c l e o t i d e  f r a c t i o n s with 7 0 $  p e r c h l o r i c a c i d a t 100C f o r 1 hour. 5. Base Composition o f Mononucleotide  Fraction  Ten absorbance u n i t s o f mononucleotide  material  i s o l a t e d from a DNA h y d r o l y s a t e weretreated with 1 ml o f 7 0 $ p e r c h l o r i c a c i d a t 100C f o r 1 hour.  The r e a c t i o n mixture was  then cooled, n e u t r a l i z e d w i t h KOH and a p p l i e d onto Whatman #1 paper.  Development o f chromatogram was e f f e c t e d by the  descending technique u s i n g Wyatt's solvent panol-HCl  (72).  UV-absorbing  system o f i s o p r o -  spots were cut out from the  paper and e l u t e d with 0.1N HC1.  The s o l u t i o n was then d r i e d  and the r e s i d u e made up to an exact volume with water.  distilled  Blank spots approximately equal i n area to the base  spots and a t equal d i s t a n c e s from the o r i g i n were c u t out and  -42s i m i l a r l y eluted. taken.  The  s p e c t r a of these s o l u t i o n s were  From the e x t i n c t i o n values known f o r these compounds,  the amount of each base present  was  r e a d i l y determined.  6. End-group A n a l y s i s of T r i n u c l e o t i d e s and To determine the 5 ' - t e r m i n a l volumes ( 2 . 5 b u f f e r , pH  ml)  8.0,  of n u c l e o t i d e  0.02M, were mixed and  nucleosides,  s o l u t i o n and  also containing MgCl  b a c t e r i a l phosphomonoesterase f o r 18 hours.  to room temperature. s i o n on a Sephadex G-25 described  heated at 100C  D e s a l t i n g was  0.02M T r i s  After  f o r 10 mins and  and  s o l u t i o n at 37C  nucleosides  nucleosides l i z e d and  0.02  Tris  moles o f MgCl2 per  liter,  f o r approximately 18 h o u r s j a f t e r which time removed by b o i l i n g and c e n t r i f u g a t i o n .  produced from the  ion-exchange chromatography. DEAE-cellulose  evaporated to  ml of snake venom phosphodiesterase  the enzyme p r o t e i n was The  exclu-  Effluent fractions  d i s s o l v e d i n 5 ml of 0.1M  which contained  t r e a t e d with 0.5  cooled  column i n a manner s i m i l a r to t h a t  r e s i d u e was  8.9,  b u f f e r , pH  incubation,  next e f f e c t e d by  f o r the d e s a l t i n g of p r o t e i n s .  The  of  with excess  c o n t a i n i n g n u c l e o t i d e m a t e r i a l were pooled and dryness.  equal  at a c o n c e n t r a t i o n  2  the mixture incubated  the r e a c t i o n mixture was  Oligonucleotides  column (1x10  The cm)  5'- nds were i s o l a t e d by e  s o l u t i o n was  run onto a  i n the c h l o r i d e form and  were e l u t e d with water.  The  e l u a t e was  the r e s i d u e d i s s o l v e d i n 1 ml of water.  the  lyophi-  -43The n u c l e o s i d e components were separated by paper chromatography.  The n u c l e o s i d e s o l u t i o n was a p p l i e d to  Whatman #1 paper and the chromatogram developed cending  technique u s i n g the water-saturated  t r a t e d NHijOH system o f H o t c h k i s s  (73).  by the des-  butanol-concen-  The n u c l e o s i d e  spots  on the paper were cut out and e x t r a c t e d with 0.1N HC1 and the e x t r a c t s were s u b j e c t e d to spectrophotometric to  analysis similar  t h a t used f o r base a n a l y s i s . To determine the 3'-terminal n u c l e o s i d e s , the o l i g o -  n u c l e o t i d e s were t r e a t e d with b a c t e r i a l phosphomonoesterase and d e s a l t e d as b e f o r e .  The r e s i d u e o b t a i n e d a f t e r  lyophi-  l i z a t i o n was d i s s o l v e d i n 5 ml o f 0.18M ammonium a c e t a t e b u f f e r , pH 5.7, which a l s o contained EDTA a t a c o n c e n t r a t i o n of  0.06M.  The s o l u t i o n was then incubated with 0.2 ml o f  spleen phosphodiesterase  a t 37C f o r 18 hours.  The n u c l e o s i d e s  were i s o l a t e d and a n a l y s e d as d e s c r i b e d b e f o r e . 7.  Separation and Analyses  of Dinucleotides  The procedure used was a v a r i a t i o n o f t h a t d e s c r i b e d by Sinsheimer  (38) f o r the s e p a r a t i o n o f mononucleotides and  d i n u c l e o t i d e s from whole DNA h y d r o l y s a t e . Dowex 1x8 i o n exchanger was used.  The r e s i n was  a l t e r n a t e l y washed with a IM HC1-1M a c e t i c a c i d  mixture  f o l l o w e d by water and then by IN NaOH u n t i l both a c i d and  _44a l k a l i n e e f f l u e n t s had no s i g n i f i c a n t a b s o r p t i o n a t 260 mu. The  cleaned r e s i n was then e q u i l i b r a t e d with a s o l u t i o n o f  0.05M ammonium a c e t a t e b u f f e r , pH 4 . 3 ,  0.015M  i n NaCl, and  then poured i n t o a column, the s i z e o f which depended upon the amount o f d i n u c l e o t i d e s to be  separated.  Immediately p r i o r to l o a d i n g , IM ammonium hydroxide was run through the column.  The d i n u c l e o t i d e s o l u t i o n was  brought to pH 1 0 with d i l u t e ammonia;,! and added to the column. E l u t i o n was s t a r t e d as d e s c r i b e d l a t e r and maintained a t a r a t e o f about 20 ml p e r hour u s i n g h y d r o s t a t i c pressure and 3-ml  f r a c t i o n s were c o l l e c t e d . For r e f r a c t i o n a t i o n , columns:d;f anal 1 bore (3 mm) were  used.  Before packing,  the i n n e r surface o f the column was  coated w i t h t r i m e t h y l s i l a n e to o b t a i n uniform through the column. a pressure  flow o f s o l v e n t  Solvent flow was e f f e c t e d by a p p l y i n g  o f 3 p . s . i . through a l a r g e syringe c y l i n d e r  which a l s o contained  the eluant  fluid.  A f t e r l o a d i n g , the column was washed with a s o l u t i o n o f 0.95M ammonium a c e t a t e b u f f e r , pH 4 . 3 and c o n t a i n i n g NaCl at a concentration o f  0.15M.  E l a t i o n was achieved  i n a step-  wise manner, u s i n g s o l u t i o n s o f 0.05M ammonium a c e t a t e j pH 4.3  c o n t a i n i n g i n c r e a s i n g amounts o f NaCl.  The d i n u c l e o t i d e s  e l u t e d were t e n t a t i v e l y i d e n t i f i e d s p e c t r o p h o t o m e t r i c a l l y by  -45measuring the A 2 6 o / 2 8 0 r a t i o o f the e f f l u e n t s a c c o r d i n g to A  the method o f Sinsheimer ( 3 8 ) . i n Table I I .  These events a r e summarized  The e l u t i o n p r o f i l e o f a t y p i c a l experiment  i s presented i n P i g . 6. Table I I .  B u f f e r s o l u t i o n s used i n the chromatography of d i n u c l e o t i d e s and the t e n t a t i v e i d e n t i f i c a t i o n o f the isomers by spectrophotometric analysis.  M o l a r i t y o f NaCl i n 0.05M ammonium acetate buffer, pH 4.3  260/ 280 of effluent  A  A  Dinucleotide isomers isolated  0.6 1.0  CC CT  1.6 1.3 1.0  AC TT  0.08  2.7 1.5 4.7 2.5  AT GT AA AG  0.2  1.6  GG  0.04 0.055 0.06  CG  I t may be noted t h a t i n both Tables I I and Fig.6, each symbol o f d i n u c l e o t i d e used i n d i c a t e s an i s o m e r i c of d i n u c l e o t i d e s . and IPC.  Thus CT r e p r e s e n t s  Dinucleotide  DNase I I h y d r o l y s i s equally w e l l .  products obtained  pair  the i s o m e r i c p a i r CT from DNase I o r  (e.g. pCpT and CpTp) were  separated  Fig. 6  Chromatography o f d i n u c l e o t i d e s on Dowex 1x8 column. D o t t e d l i n e s i n d i c a t e m o l a r i t y o f NaCl i n e l u a n t liquid. Three-ml f r a c t i o n s were c o l l e c t e d .  -47It  can be seen from F i g . 6 that under the c o n d i t i o n s  used, the d i n u c l e o t i d e p a i r GT was not separated Therefore  the f r a c t i o n s comprising  from AA.  t h i s peak were pooled, made  a l k a l i n e with NH^OH and readsorbed onto another column o f Dowex 1 x 8 ( 0 . 3 x 2 0 cm).  Use o f 0.2M ammonium a c e t a t e  pH 3 . 7 » c o n t a i n i n g 0.08 moles o f NaCl p e r l i t e r  buffer,  then cleanly-  r e s o l v e d the two components. Each i s o m e r i c p a i r o f d i n u c l e o t i d e s was r e s o l v e d i n t o i t s components a c c o r d i n g Garilhe et a l . ( 7 4 ) .  to a method d e s c r i b e d by de  The f r a c t i o n s comprising  t i d e p a i r were pooled and l y o p h i l i z e d .  one d i n u c l e o -  The r e s i d u e was  d i s s o l v e d i n 1 ml o f 0.01M T r i s b u f f e r , pH 8.0, which a l s o contained  MgC^ a t a c o n c e n t r a t i o n  b a c t e r i a l phosphomonoesterase.  o f 0.01M and t r e a t e d w i t h  The s o l u t i o n was then l y o -  p h i l i z e d , r e d i s s o l v e d i n water and a p p l i e d q u a n t i t a t i v e l y as a streak to Whatman #3 f i l t e r paper.  Hotchkiss's  system ( 7 3 ) was used to develop the chromatogram. upon the Rf values o f the n u c l e o s i d e  solvent  Depending  components present, the  d u r a t i o n o f development was v a r i e d from 6 to 14 days.  Thus,  the d i n u c l e o t i d e s AC, AT and CT were i r r i g a t e d f o r 6 days; d i n u c l e o t i d e s c o n t a i n i n g G were separated p a i r s a f t e r 14 days' development. obtained  i n t o the i s o m e r i c  UV-absorbing spots  were e l u t e d with 0.1N HC1.  thus  The i d e n t i t y o f each  spot was determined as d e s c r i b e d f o r the end-group  analyses  -48of t r i n u c l e o t i d e s . 8.  Denaturation o f C a l f Thymus DNA One hundred mg o f c a l f thymus DNA was d i s s o l v e d i n  0.02M NaCl.  The r e s u l t i n g s o l u t i o n was heated a t 100C f o r  10 mins and r a p i d l y c o o l e d by immersing the v e s s e l i n i c e water.  To t h i s s o l u t i o n o f heated DNA, an equal volume o f  0.2M ammonium a c e t a t e , 0.06M with r e s p e c t to MgCl2 was added and the pH a d j u s t e d to 6 . 8 .  T h i s was used as s u b s t r a t e s o l u -  t i o n f o r i n t e s t i n a l DNase I . 9.  Shearing o f C a l f Thymus DNA As d i s c u s s e d l a t e r , h e a t i n g o f DNA as d e s c r i b e d  above d i d not denature  the DNA completely.  which might be more e a s i l y denatured, mus DNA  To o b t a i n m a t e r i a l  a solution of calf  thy-  (250 ug per ml i n 0.02M NaCl) was s u b j e c t e d to mech-  a n i c a l shearing i n the S e r v e l l Omni Mixer.  Three d i f f e r e n t  samples were sheared a t 2,000 r.p.m. f o r 15 sec, 16,000 r.p.m. f o r 1 min and 16,000 r.p.m. f o r 5 mins.  Afterwards, the  s o l u t i o n s were d i l u t e d with 0.02M NaCl to a f i n a l t i o n o f 100 ug DNA p e r ml and heat-denatured  concentra-  as b e f o r e .  RESULTS AND DISCUSSION The l i n k a g e s p e c i f i c i t i e s o f the two DNases from the i n t e s t i n a l mucosa o f the r a t were s t u d i e d with the view  -49to u s i n g them as p o s s i b l e reagents  f o r the d e t e r m i n a t i o n o f  n u c l e i c a c i d s t r u c t u r e and n u c l e o t i d e sequence. experiments i n these  Certain  s t u d i e s (see l a t e r ) showed t h a t DNase I  was a c t i v a t e d to d i f f e r e n t extents by M g same enzyme was a c t i v e towards heated s i d e r a b l y l e s s potency.  Specificity  ++  and M n  s t u d i e s with t h i s  c o n d i t i o n s : n a t i v e DNA as s u b s t r a t e , M g DNA as s u b s t r a t e , M g  . The  DNA, though with con-  were t h e r e f o r e c a r r i e d out under three d i f f e r e n t  and heated  ++  ++  or Mn  enzyme  sets of ++  as a c t i v a t o r  as a c t i v a t o r .  ++  Heat d e n a t u r a t i o n o f c a l f thymus DNA was performed a c c o r d i n g to accepted procedures m e l t i n g p r o f i l e o f the heated  (27).  However, when the  sample was examined, the DNA was  found to s t i l l possess a c o n s i d e r a b l e amount o f secondary s t r u c t u r e , showing one d i f f u s e Tm a t about 53C and another at 87C  Shearing o f DNA even a t 16,000 r.p.m. f o r 5 minutes  p r i o r to h e a t i n g d i d not completely strandedness  o f the DNA.  a b o l i s h the double-  The m e l t i n g p r o f i l e s o f the v a r i o u s  DNA samples a r e shown i n F i g . 7.  More vigorous shearing was  not attempted f o r f e a r o f i n t r o d u c i n g spurious end groups. As p o i n t e d out by L i n and Chargaff evidence  t h a t denatured  of segregated  (75),  there i s no s t r i c t  p r e p a r a t i o n s o f DNA c o n s i s t e d e n t i r e l y  complementary s t r a n d s .  In a l l probability,  such p r e p a r a t i o n s contained a tangle o f p a r t i a l l y f r a y e d o r b u l g i n g , but never completely  separated,  complementary  50.  l.4r  TEMPERATURE Fig. ?  °C  Melting p r o f i l e s of variously treated calf thymus DNA. Cone, o f DNA i n these e x p e r i ments vias 50 ^5 P ml. Determinations were made i n O.IK ammonium a c e t a t e b u f f e r , pH 6 . 8 - 0 . 0 3 K K g C l - 0 . 0 1 M NaCl. e r  2  -51structures. DNA  Since the i n t e s t i n a l DNase I a t t a c k e d n a t i v e  about 5 to 10  times f a s t e r than heated DNA,  i t was  not  c l e a r whether the a c t i v i t y o f the enzyme towards heated was  not due  to the presence of double-stranded  DNA  regions i n  the heated sample. An attempt was  made to o b t a i n s i n g l e - s t r a n d e d  DNA  by f r a c t i o n a t i o n of a heated sample on MAK  columns a c c o r d i n g  to the method of Mandell and Hershey ( 7 6 ) ,  but the  was  too low  to be of p r a c t i c a l  The  use.  problem of l i n k a g e s p e c i f i c i t y was  a) examining the base composition end group a n a l y s e s o f the t r i and  c) determining  tides. follows.  The  t a c k l e d by  of the mononucleotides,  dinucleo-  these kinds of a n a l y s e s i s as  Suppose the o l i g o n u c l e o t i d e s show a preponderance  of pyrimidines  (Py) a t the 5 ' - t e r m i n i , i t would be assumed  t h a t the l i n k a g e s of the type pXpPy i n the DNA  are more  s e n s i t i v e to the enzyme than the other l i n k a g e s . a preponderance of p u r i n e s  of the 16  hydrolysate,  Similarly,  (Pu) a t the 3 ' - e n d s would i n d i -  cate an enzymatic preference any  b)  and higher o l i g o n u c l e o t i d e s  the r e l a t i v e f r e q u e n c i e s of the  reasoning behind  yield  f o r the pPupX bond.  Also, i f  p o s s i b l e d i n u c l e o t i d e s were absent from the i t would be i n f e r r e d t h a t the l i n k a g e r e p r e -  sented by t h i s p a r t i c u l a r d i n u c l e o t i d e i s p r e f e r e n t i a l l y susceptible.  -52However, these assumptions are v a l i d only i f the same type o f l i n k a g e i s p r e f e r e n t i a l l y h y d r o l y s e d the course  o f the r e a c t i o n .  throughout  Vanecko and Laskowski (77),  working with p a n c r e a t i c DNase I, found t h a t as enzymatic d i g e s t i o n proceeded, s m a l l e r molecules were produced which were p r o g r e s s i v e l y r e s i s t a n t to a t t a c k .  The r a t e o f the  r e a c t i o n slowed down c o n s i d e r a b l y and c o u l d be r e s t o r e d only by the a d d i t i o n o f a very l a r g e amount o f enzyme.  The same  phenomenon o f a u t o r e t a r d a t i o n was observed with the i n t e s t i n a l enzyme.  One e x p l a n a t i o n f o r a u t o r e t a r d a t i o n i s t h a t the  s m a l l e r products  are r e s i s t a n t to enzymatic a c t i o n and may  a c t u a l l y serve as c o m p e t i t i v e l i n h i b i t o r s o f the s u b s t r a t e . Thus a p o t e n t i a l l y  s u s c e p t i b l e l i n k a g e may not be a c t e d upon  i f i t i s present w i t h i n a small o l i g o n u c l e o t i d e . A f u r t h e r c o m p l i c a t i o n i n the study o f l i n k a g e s p e c i f i c i t y i s the f i n d i n g t h a t the p r o x i m i t y o f the monoe s t e r i f i e d phosphate group i n f l u e n c e d the a c t i v i t y o f pancr e a t i c DNase I .  Vanecko and Laskowski ( ? 8 )  subjected  frag-  ments o b t a i n e d from a DNase I I d i g e s t to the a c t i o n o f p a n c r e a t i c DNase I and found t h a t while no n u c l e o s i d e s o r mononucleotides were formed i n the h y d r o l y s a t e ,  3*»5*-mono-  n u c l e o s i d e diphosphates were abundant and were found e a r l y i n the course  of r e a c t i o n .  T h i s was i n t e r p r e t e d to i n d i c a t e  that the p r o x i m i t y o f the monophosphate l a b i l i z e d the  -53preceding i n t e m u c l e o t i d e linkage. not  While such s t u d i e s have  been c a r r i e d out with the i n t e s t i n a l  enzyme, the p o s s i -  b i l i t y o f l a b i l i z a t i o n o f a pho)sphodiester bond due to a nearby phosphomonoester group cannot be d i s r e g a r d e d . Ralph et a l . ( 7 9 ) i n d i c a t e d t h a t s i n c e a t e t r a n u c l e o t i d e appeared to be the s m a l l e s t o l i g o n u c l e o t i d e capable of being a t t a c k e d , i t may be erroneous to d i s c u s s the mode of a c t i o n o f endonucleases i n terms o f s i n g l e i n t e m u c l e o t i d e bonds; the s p e c i f i c i t y o f such enzymes may be d i c t a t e d by a sequence  o f bases i n the v i c i n i t y o f the bond c l e a v e d .  Furthermore,  some authors ( 5 6 , 80) noted t h a t the s p e c i f i c i t y  of p a n c r e a t i c DNase I depended upon the nature o f the i o n s present. of  For example, Bollum  ( 5 6 ) prepared polymers  composed  complementary homopolymeric chains such as dl-dC and  s u b j e c t e d them to the a c t i o n o f p a n c r e a t i c DNase I . When Mg^was used as the a c t i v a t i n g i o n , o n l y the d l c h a i n was d i g e s t e d . 1  The a d d i t i o n o f C a Mg  + +  + +  (0.002M) t o a medium c o n t a i n i n g 0.01M  r e s u l t e d i n the d i g e s t i o n o f dC as w e l l as d l .  and Khorana ( 7 9 ) found t h a t the o l i g o n u c l e o t i d e  Ralph  hexaadenylate  was d i g e s t e d by DNase I f a s t e r than hexathymidylate i n the presence o f M n . ++  I n the presence o f M g , Bollum ++  ( 5 6 ) found  t h a t the d i g e s t i o n o f p o l y dA:dT d i d not proceed u n i f o r m l y and t h a t fragments o f o l i g o a d e n y l a t e remaining i n the d i g e s t were l a r g e r than the fragments o f o l i g o t h y m i d y l a t e .  These  -54two  experiments suggested t h a t the i o n i c  i n f l u e n c e s the a f f i n i t y of the  substrates  environment even though they  are both s u s c e p t i b l e i n the presence of e i t h e r i o n . Spleen DNase I I was  a l s o found by Koerner and  sheimer (9) to undergo a continuous a u t o r e t a r d a t i o n the  same way  f o r e be  as p a n c r e a t i c  DNase I .  Thus i f a d i n u c l e o t i d e XY  which i t r e p r e s e n t s  s p e c i f i c i t y of  i s missing  The  from  the  the  concluded that the  i s preferentially  a t the l e v e l of d i n u c l e o t i d e s . be  there-  imposed upon the p r e v i o u s g e n e r a l i z a t i o n concerning  p r o d u c t s of h y d r o l y s i s , i t might be age  i n much  L i m i t a t i o n s must  the f i n a l p r o d u c t s of the r e a c t i o n and enzyme.  Sin-  cleaved,  but  only  same c o n c l u s i o n may  not  extended to e a r l y c l e a v a g e s . With the r e s e r v a t i o n i n mind that c o n c l u s i o n s  garding linkage  conditions  stage of an apparent e q u i l i b r i u m and  (pH,  with the i n t e s t i n a l  ionic  DNases are examined.  frequencies  neighbour f r e q u e n c i e s c a l f thymus DNA  under  environment) of study, data o b t a i n e d  summarized i n Tables I I I through VI. dinucleotide  re-  s p e c i f i c i t y drawn from product a n a l y s e s are  v a l i d only a t the the  link-  These data are  In Table IV, where the  are l i s t e d , the data on  nearest  o b t a i n e d by Koenberg et a l . (81)  are i n c l u d e d as a b a s i s f o r comparison.  for  -55Table I I I .  Percentage base composition o f mononucleotides i n DNA h y d r o l y s a t e s .  Condition of hydrolysis  A  C  G  T  Native DNA, Mg , DNase I  13.8  8.4  8.6  69.2  Native DNA, Mn , DNase I  21.5  18.7  12.3  47.5  Heated DNA, M g , DNase I  18.0  13.8  10.4  57.8  Native DNA, DNase I I  27.7  20.2  21.9  30.2  ++  ++  ++  -56Table IV.  Percentage f r e q u e n c i e s o f d i n u c l e o t i d e s i s o l a t e d from DNA h y d r o l y s a t e s o b t a i n e d under v a r i o u s r e a c t i o n c o n d i t i o n s .  Dinucleotide  Nearest neighbour frequency (81)  Native DNA, Mg++, DNase I  Native DNA, Mn , DNase I ++  Heated DNA, Mg , DNase I ++  Native DNA, DNase I I  AA  8.9  3.3  8.2  8.5  16.2  AC  5.2  0  3.8  4.32  0  AG  7.2  3.3  5.2  9.21  6.4  AT  7.3  0  3.1  2.15  10.1  CA  8.0  20.7  6.3  4.04  5.2  4.8  9.5  4.17  10.2  CC CG  1.6  1.9  2.9  1.25  7.1  CT  5.83  10.4  7.7  6.53  6.5  GA  6.4  6.0  5.7  13.21  5.5  GC  4.4  8.1  8.4  4.57  0  GG  5.0  4.0  3.2  8.53  8.4  GT  5.6  0  8.7  3.71  1.0  TA  5.3  10.2  5.7  8.82  3.1  TC  6.7  4.5  8.0  3.61  5.7  TG  7.6  15.6  4,. 4  8.17  3.5  TT  8.7  7.2  9.2  9.21  11.2  -57Table V a .  Percentage base f r e q u e n c i e s a t the 5 ' - t e r m i n i of t r i n u c l e o t i d e s i n DNA h y d r o l y s a t e s  C o n d i t i o n s <of hydrolysis  A?  C  G  T  Native DNA, Mg++, DNase I  20.3  28.1  15.7  35.9  Native DNA, Mn , DNase I  20.5  28.2  20.3  31.0  Heated DNA, M g , DNase I  10  16.2  37.4  36.4  Native DNA, DNase I I  14.7  32.9  27.6  24.8  ++  ++  Table V b.  Percentage base f r e q u e n c i e s a t the 3 ' - t e r m i n i o f t r i n u c l e o t i d e s i n DNA h y d r o l y s a t e s  C o n d i t i o n s <Df hydrolysis  A*  C  G  T  Native DNA, M g , DNase I  27.6  24.4  23.5  24.5  Native DNA, M n , DNase I  27.5  24.7  20.3  27.5  Heated DNA, M g , DNase I  8.8  34.1  18.6  38.5  Native DNA, DNase I I  17.4  17.3  34.9  30.4  ++  ++  ++  * The commercial p r e p a r a t i o n o f spleen phosphodiesterase was found to be contaminated with a deaminase a c t i v e towards adenine d e r i v a t i v e s . The V a l u e s f o r adenine r e p o r t e d here a r e t t o s e o b t a i n e d f o r i n o s i n e formed from adenosine by the deaminase a c t i v i t y .  -58Table VI a.  Percentage base f r e q u e n c i e s a t the 5 ' - t e r m i n i of o l i g o n u c l e o t i d e s i n DNA h y d r o l y s a t e s  Conditions o f hydrolysis  A  C  Native DNA, M g , DNase I  20.5  Native DNA, Mn , DNase I  G  T  27.9  14.6  37.0  23.5  27.5  22.6  26.4  Heated DNA, M g , DNase I  18.6  29.8  18.6  33.0  Native DNA, DNase I I  15.5  29.2  30.6  24.7  ++  ++  ++  Table VI b.  Percentage base f r e q u e n c i e s a t the 3 * - t e r m i n i o f o l i g o n u c l e o t i d e s i n DNA h y d r o l y s a t e s  Conditions of hydrolysis  A*  C  G  Native DNA, M g , DNase I  36.1  23.1  22.5  18.3  Native DNA, M n ± , DNase I  29.4  23.1  21.1  26.4  Heated DNA, M g , DNase I  15.3  46.8  10.4  27.5  17.6  14.5  37.7  30.2  ++  +  ++  Native DNA, DNase I I  * See footnote f o r Table Vb.  T  - 59-  Th e most s t r i k i n g f e a t u r e i n the i n t e s t i n a l DNase I r e a c t i o n i s the preponderance o f t h y m i d y l i c a c i d among the mononucleotide p r o d u c t s under a l l c o n d i t i o n s s t u d i e d .  In  the presence o f M g , t h y m i d y l i c a c i d c o n s t i t u t e s 60 to 70% ++  o f a l l the mononucleotides.  This i s reminiscent  DNase I ( 3 8 ) .  o f mononucleotides i m p l i e s a  The formation  c e r t a i n k i n d o f exonuclease a c t i v i t y .  of pancreatic  I t might a l s o be  i n f e r r e d t h a t the mononucleotides were formed from s i n g l e stranded  o l i g o n u c l e o t i d e s , otherwise the mononucleotide  n y l i c a c i d would be as abundant  as t h y m i d y l i c a c i d .  ade-  Since  the l i n k a g e s AT and TA are q u i t e common i n c a l f thymus DNA as judged from the n e a r e s t  neighbour frequency a n a l y s e s o f  Josse e t a l . ( 8 1 ) , the mononucleotides must be formed a s p e c i f i c end o f the o l i g o n u c l e o t i d e s .  Whether the 3'-ends  o r 5'-ends f u r n i s h e d the mononucleotides i s not c l e a r . very  from  One  s l i g h t c l u e stems from the f a c t t h a t the 3'-ends o f the  oligonucleotides  (Table VI b) o b t a i n e d  the presence o f M g  + +  w i t h n a t i v e DNA i n  bear r e l a t i v e l y few thymine  residues,  i n d i c a t i n g that the t h y m i d y l i c a c i d s might have a r i s e n from t h i s end. midylate  In addition  . the high percentage (37%) o f thy-  a t the 5'-ends o f the o l i g o n u c l e o t i d e s would seem  to suggest t h a t the 3'-ends a r e the o r i g i n o f mononucleotides. Hanson ( 8 2 ) suggested the p o s s i b i l i t y  t h a t enzymes  -60may  a c t a g a i n on a s u b s t r a t e by a repeated a t t a c k  before  d i s s o c i a t i o n from the cleaved  substrate.  process Hurst  (80)  suggested that the exonuclease a c t i v i t y of DNases might  be  a r e s u l t of t h i s m u l t i p l e a t t a c k mechanism.  Based on h i s  data f o r the h y d r o l y s i s of DNA  DNase I, i n  by p a n c r e a t i c  which he found that the c o n c e n t r a t i o n pTpTpT ( 0 . 5 1 $ ) was dom  much lower than that c a l c u l a t e d f o r a  d i s t r i b u t i o n (2.18$) and  about one  of the t r i n u c l e o t i d e  t h i r d of the  sum  the amount of thymidylate  of pT and pTpT, he  ran-  was  suggested that  t h y m i d y l i c a c i d might have been formed by f u r t h e r h y d r o l y s i s of the t r i n u c l e o t i d e pTpTpT.  However, from h i s data, he  unable to e s t a b l i s h which end of the t r i n u c l e o t i d e had  was  given  r i s e to the mononucleotides. The seem to apply  mechanism suggested by Hurst to the i n t e s t i n a l DNase I .  (80)  does not  Table VII l i s t s  the  amount o f o l i g o n u c l e o t i d e phosphorus as a percentage of  total  phosphorus.  and  IV,  i t can be  When viewed i n c o n j u n c t i o n  with Tables I I I  seen that the amount of mononucleotide thymi-  d y l i c a c i d f a r exceeds that of the d i n u c l e o t i d e pTpT i n the Mg++-catalysed DNA One  hydrolysate.  f a c t which argues a g a i n s t the formation  of  mononucleotides from the 3'-ends of o l i g o n u c l e o t i d e s i s that among the d i n u c l e o t i d e s , l i n k a g e s of the type pTpX c o n s t i t u t e more than 35$ o f the t o t a l .  Since pTpX l i n k a g e s must  -6l-  f i r s t be h y d r o l y s e d  to f u r n i s h o l i g o n u c l e o t i d e s  a c i d a t the 3 ' - t e r m i n i  thymidylic  which then serve as subs-  t r a t e s f o r the exonuclease a c t i v i t y ,  the h i g h l e v e l o f pTpX  d i n u c l e o t i d e s would seem to argue a g a i n s t l y s e d during out  the course o f r e a c t i o n .  e a r l i e r , and a l s o d i s c u s s e d  t h e i r being hydro-  However, as p o i n t e d  by Laskowski  (1),  t i d e s a r e probably formed r e l a t i v e l y l a t e during and  bearing  dinucleothe r e a c t i o n ,  do not n e c e s s a r i l y r e f l e c t the s p e c i f i c i t y o f the enzyme  during  e a r l y cleavage.  Table VII Percentage o f o l i g o n u c l e o t i d e as compared to t o t a l  Conditions o f hydrolysis  Mononucleotide  N a t i v e DNA, M g , DNase I  1.23  N a t i v e DNA, Mn , DNase I  4.4  ++  ++  Heated DNA, Mg , DNase I Native DNA, DNase I I  phosphorus  ©inucleo- T r i n u oligocleotide nucleotide tide 12.0  85.58  40.8  26.4  29.4  2.2  6.8  20.5  71.5  4.25  4.15  8.75  82.85  In c o n t r a s t  1.19  to p a n c r e a t i c  DNase I , thea c t i o n o f  which was shown by Sinsheimer (38) to produce  dinucleotides  which comprise about \7% o f the t o t a l DNA phosphorus when Mg  ++  was the a c t i v a t i n g i o n , the i n t e s t i n a l DNase I r e l e a s e d  only 1 . 2 $ o f the t o t a l DNA phosphorus a s d i n u c l e o t i d e s .  Of  -62the 16 p o s s i b l e major d i n u c l e o t i d e s , three were m i s s i n g from the i n t e s t i n a l DNase I d i g e s t o f DNAj these were pApT, pApC and pGpT.  I t seemed to be more than c o i n c i d e n c e t h a t  these three d i n u c l e o t i d e s were n o t found. polarity  Assuming the a n t i -  o f the strands i n the DNA molecule,  the three l i n k -  ages AT, AC and GT would be p l a c e d o p p o s i t e each o t h e r i n ...pApT....  ...pApC...  the manner ....TpAp... and ....TpGp...  .  I f i t i s assumed  t h a t these three l i n k a g e s were h y d r o l y s e d u n i f o r m l y  through-  out the course o f enzyme a c t i o n , there would be strong to b e l i e v e t h a t the bonds I^Atwo-strand  approximately  n  d  ~TG~  w  e  r  e  c l e a v e d by a  Thomas (83) found by t i t r i m e t r y  scission.  light-scattering  a  reason  and  t h a t p a n c r e a t i c DNase I must h y d r o l y s e  200 phosphodiester  bonds before the weight  average molecular weight o f DNA was decreased by a f a c t o r o f 2.  Young and Sinsheimer  biological  activity  (84) concluded  from the l o s s o f  (immunity o f phage and mottled and c l e a r  plaque) when \-phage DNA was a c t e d upon by p a n c r e a t i c DNase I t h a t the enzyme c l e a v e d a t random and on an average about f o u r s i n g l e - s t r a n d e d cleavages o c c u r r e d before i n f e c t i v i t y of the phage was l o s t . t h a t double-stranded theless occurred.  Both experiments p o i n t e d to the f a c t  s c i s s i o n s were r e l a t i v e l y r a r e but never-  I f the i n t e s t i n a l enzyme a l s o a t t a c k e d  both strands o f DNA a t the same l e v e l , the most probable s i t e s were a t the pApT, pApC and pGpT bonds.  -63In support  o f the c o n t e n t i o n t h a t the three  link-  ages i n q u e s t i o n may indeed be the p r e f e r r e d s i t e s o f i n c i s i o n i s the f a c t t h a t both T and C a r e abundant i n the 5'-ends o f the t r i - and o l i g o n u c l e o t i d e s i s o l a t e d from the h y d r o l y s a t e , the two c o n s t i t u t i n g about 6 5 $ o f the t o t a l . While one must be c a u t i o u s i n attempting l i n k a g e s p e c i f i c i t y from product  to g e n e r a l i z e about  analyses,  seems to l e a n i n favour o f the p r e f e r e n c e  the evidence o f the i n t e s t i n a l  DNase I f o r the three l i n k a g e s AT, AC and GT. That t h i s s p e c i f i c i t y i s not a b s o l u t e c l e a r l y seen from these a n a l y s e s .  i s also  Thus the d i n u c l e o t i d e s AA  and AG were both found to be present  i n much s m a l l e r amounts  than i n d i c a t e d by the nearest neighbour frequency suggesting Looking  studies,  a l a b i l i t y o f these l i n k a g e s towards the enzyme.  a t the matter from another angle,  the f a c t t h a t the  amount o f the two d i n u c l e o t i d e s pCpA and pTpG f a r exceeded the l e v e l o f the r e s p e c t i v e l i n k a g e s i n n a t i v e DNA can only be  e x p l a i n e d by assuming t h a t a l l the other l i n k a g e s were  hydrolysed  with g r e a t e r ease. When manganese i o n s were used as the a c t i v a t i n g i o n ,  the products when M g  ++  o f h y d r o l y s i s were q u i t e d i f f e r e n t from  those  was used, both with r e s p e c t to s i z e and nature.  Most s t r i k i n g was a c o n s i d e r a b l e i n c r e a s e i n the amount o f  -64(40.8$  dinucleotid.es Once again,  o f t o t a l o l i g o n u c l e o t i d e s ) (Table V I I ) .  t h y m i d y l i c a c i d was the most frequent  component  o f the mononucleotides, i n d i c a t i n g that the mechanism o f formation  o f mononucleotides was probably the same with  either ion. not  However, the preponderance o f thymidylate  so overwhelming as with the case o f M g . ++  was  The l e v e l s o f  the other three mononucleotides were a l s o r a i s e d , adenylate notably  so.  The s i g n i f i c a n c e o f t h i s w i l l be d i s c u s s e d  later.  Among the products o f the M n - a c t i v a t e d DNase I + +  r e a c t i o n , a l l 16 p o s s i b l e d i n u c l e o t i d e s were found.  The  p a t t e r n o f t h e i r d i s t r i b u t i o n was a l s o shown to resemble t o a c e r t a i n degree the nearest  neighbour frequency  pattern  (Table I V ) . T h i s i s i n t e r p r e t e d as meaning that the i n t e s t i n a l enzyme a t t a c k e d n a t i v e DNA i n a random manner when a c t i v a t e d by manganese. Becking and Hurst a c t i v a t i o n of pancreatic f r a c t i o n represented  (80), studying  the e f f e c t o f M n  DNase I , found that the d i n u c l e o t i d e  about 5°$ o f the t o t a l phosphate. But  among t h i s f r a c t i o n , the three d i n u c l e o t i d e s pApG, pGpA and pGpG were missing,  i n d i c a t i n g a p r e f e r e n t i a l a t t a c k by the  enzyme on the o l i g o n u c l e o t i d e s , though the r e s u l t s were not conclusive.  In t h i s respect,  DNases d i f f e r .  ++  the i n t e s t i n a l and p a n c r e a t i c  -65The r e s u l t s o f end-group a n a l y s e s o f the t r i n u c l e o t i d e s and o l i g o n u c l e o t i d e s (Tables V and VI) a l s o p o i n t e d s t r o n g l y to a random a t t a c k .  The d i f f e r e n c e i n a c t i o n be-  tween i n t e s t i n a l DNase I and p a n c r e a t i c DNase I was a g a i n evident.  Hurst  the presence  ( 5 5 ) found t h a t with l i m i t e d h y d r o l y s i s i n  o f Mn , p a n c r e a t i c DNase I gave r i s e to o l i g o ++  n u c l e o t i d e s b e a r i n g twice as many p y r i m i d i n e r e s i d u e s as p u r i n e s a t the 5'-ends, i . e . , the A+G/C+T r a t i o was about 0.5.  A f t e r exhaustive d i g e s t i o n , t h i s r a t i o i n c r e a s e d to  0.6.  He concluded  t h a t with Mn , pXpPy l i n k a g e s were c l e a ++  ved e a r l y i n the r e a c t i o n ; l i n k a g e s o f the type pXpPu were h y d r o l y s e d during the l a t e r stages.  Table VI shows t h a t with  the i n t e s t i n a l enzyme, the Pu/Py r a t i o a t the 5 ' - t e r m i n a l s of  the o l i g o n u c l e o t i d e s i s 0 . 8 5 ,  p o i n t i n g to random a t t a c k  with perhaps a s l i g h t p r e f e r e n c e towards pXpPy bonds. The Pu/Py r a t i o f o r the t r i n u c l e o t i d e s  (Table V) i s 0 . 6 9 .  On a  s t a t i s t i c a l b a s i s , i t i s l o g i c a l to assume t h a t the t r i n u c l e o t i d e s were formed r e l a t i v e l y l a t e r than the h i g h e r o l i g o nucleotides.  On t h i s assumption, i t would seem t h a t the  p r e f e r e n c e o f the enzyme f o r pXpPy bonds became more pronounced at  the l a t e r stages o f h y d r o l y s i s . Measurements on the i n i t i a l i n c r e a s e i n absorbance  of  a DNA s o l u t i o n t r e a t e d with i n t e s t i n a l DNase I showed  that the v e l o c i t y was much h i g h e r when M n  + +  was used as  -66a c t i v a t i n g i o n (see S e c t i o n I V ) .  Since more bonds were  broken p e r u n i t time with Mn , the decrease i n s i z e o f the ++  o l i g o n u c l e o t i d e s became more r a p i d .  Chances were that some  p o t e n t i a l l y more s u s c e p t i b l e l i n k a g e s (pApC, pApT and pGpT) were not a t t a c k e d e a r l y due to t h e i r p o s i t i o n i n the i n t e r i o r of a small molecule.  The mode o f a t t a c k would be random a t  the e a r l y stage o f the r e a c t i o n . the  At the l a t e r stages, however,  "normal" order o f p r e f e r e n c e was r e s t o r e d , as manifested  i n more pXpPy l i n k a g e s being T h i s evidence  hydrolysed.  p o i n t s to a d i f f e r e n c e i n i n i t i a l  a c t i o n o f the i n t e s t i n a l enzyme when M n activator.  The decrease i n thymidylate  ++  or Mg  content  ++  was used as  o f the mono-  n u c l e o t i d e f r a c t i o n c o u l d be e x p l a i n e d by a reduced product i o n o f s u b s t r a t e s c a r r y i n g the necessary p a r t o f the M n - a c t i v a t e d ++  end groups on the  enzyme.  When heated DNA was used as the s u b s t r a t e f o r i n t e s t i n a l DNase I, the i n i t i a l  r a t e o f the r e a c t i o n was  reduced t o l e s s than 20$ o f t h a t when n a t i v e DNA was used. Two p o s s i b l e reasons f o r t h i s must be c o n s i d e r e d .  The f i r s t  i s t h a t the i n t r i n s i c r a t e o f a t t a c k by the enzyme on s i n g l e stranded  DNA I s much l e s s than that with n a t i v e DNA.  The  other, e q u a l l y p l a u s i b l e , i s t h a t s i n c e h e a t i n g f a i l e d to completely  d i s s o c i a t e the two strands o f the molecule, the  -67-  slower r a t e may  have been due  of double-stranded r e g i o n s .  to a reduced c o n c e n t r a t i o n However, i f such were the case,  the r e s u l t i n g products should resemble native substrate.  those formed from a  As can be seen from Tables IV to VI, with  the e x c e p t i o n of the mononucleotide  fraction,  such i s not  the case. Table IV shows tohat when heated DNA  was  hydrolysed  by DNase I i n the presence of M g , a l l types of d i n u c l e o t i d e s ++  were formed. up about  However, d i n u c l e o t i d e s of the type pXpPu made  65% of the t o t a l f r a c t i o n ,  probably p r e f e r r e d the pXpPy bonds.  suggesting that the enzyme The r e l a t i v e abundance  o f C and T a t the 5'-ends o f the o l i g o n u c l e o t i d e s may c i t e d i n support o f t h i s .  be  R e s u l t s o b t a i n e d with the t r i n u -  c l e o t i d e s were l e s s c o n c l u s i v e . The a c t i o n of i n t e s t i n a l DNase I on heated DNA resembles  thus  the a c t i o n of the enzyme a t the l a t e r stages of i t s  a t t a c k on n a t i v e DNA.  T h i s i s not a t a l l s u r p r i s i n g s i n c e  i n a l l p r o b a b i l i t y the o l i g o n u c l e o t i d e s u b s t r a t e s a v a i l a b l e then were i n the s i n g l e - s t r a n d e d form. r e a t i c DNase I on heated DNA comparison  The a c t i o n of panc-  has not been r e p o r t e d , and no  with the i n t e s t i n a l enzyme can be made.  I t would  be of i n t e r e s t to know i f the argument presented above a l s o a p p l i e s to the p a n c r e a t i c enzyme.  -68When i n t e s t i n a l  DNase I I was used to degrade  DNA,  the mononucleotides o b t a i n e d d i d not show any unusual f e a ture.  No d e f i n i t i v e  origin.  c o n c l u s i o n c o u l d be drawn as to t h e i r  I n view o f t h i s u n c e r t a i n t y , the s i g n i f i c a n c e o f the  end groups i n the t r i - and o l i g o n u c l e o t i d e s c o u l d not be assessed with confidence,  s i n c e they may i n a c t u a l f a c t  r e f l e c t the s p e c i f i c i t y o f the exonuclease a c t i v i t y o f the enzyme.  Thus, whereas the end groups i n d i c a t e d the l i n k a g e s  GpGp, GpGp, TpCp and TpGp to be most s u s c e p t i b l e , the d i n u c l e o t i d e s tended to suggest t h a t the l i n k a g e s ApCp, GpCp and GpTp t o be the most v u l n e r a b l e .  I t may be noted t h a t ApCp  and GpTp are s i t u a t e d d i r e c t l y  opposite  double-stranded  The l i n k a g e pGpC i s a l s o p l a c e d  DNA molecule.  o p p o s i t e pGpC bond a c r o s s the s t r a n d s .  each o t h e r on the  I f these were indeed  the p r e f e r r e d s i t e o f a t t a c k , a double-stranded  scission  appears to be i n d i c a t e d . Sinsheimer (9)  s t u d i e d the a c t i o n o f s p l e n i c DNase  I I and f a i l e d to d e t e c t any l i n k a g e s p e c i f i c i t y by end group analyses.  Vanecko and Laskowski  enzyme a t three d i f f e r e n t  (85)  i n v e s t i g a t e d the same  stages o f r e a c t i o n and n o t i c e d that  the s p e c i f i c i t y o f the enzyme changed as the r e a c t i o n progressed.  They (85)  concluded  t h a t GpCp was the most l i k e l y  t a r g e t o f a t t a c k d u r i n g the e a r l i e s t  stages o f the r e a c t i o n .  -69-  T h i s c o n c l u s i o n was  based on end-group a n a l y s e s , and,  d i s c u s s e d above, c o u l d not be d e s c r i b e d as The  unequivocal.  only c o n c l u s i o n which can be drawn from  s t u d i e s c a r r i e d out on the r a t i n t e s t i n a l  as  these  deoxyribonucleases  i s t h a t no c l e a r - c u t o r d e r o f s p e c i f i c i t y c o u l d be demonstrated. was  A c e r t a i n degree of p r e f e r e n c e  f o r some l i n k a g e s  observed, but the v a l i d i t y even of t h i s i s q u e s t i o n a b l e  i n view of the e f f e c t of a u t o r e t a r d a t i o n and other T h i s i s not e n t i r e l y unexpected.  factors.  Since there are i n g e n e r a l  o n l y f o u r bases w i t h i n a p o l y n u c l e o t i d e , i t i s d i f f i c u l t imagine how  to  an enzyme can be l i m i t e d i n i t s a t t a c k to only  a few s i t e s on the molecule by a simple base  specificity.  C l e a r l y , i t would have to possess the c a p a c i t y to  recognize  long sequences of r e s i d u e s along the p o l y n u c l e o t i d e chain, a s p e c i f i c i t y i t would be extremely d i f f i c u l t with the methods now  available.  to demonstrate  -70IV.  KINETIC STUDIES OF THE INTESTINAL DEOXYRIBONUCLEASE I REACTION  EXPERIMENTAL 1.  Measurement o f DNase  Activity-  Enzyme a c t i v i t y was measured by Kunitz* s s p e c t r o photometric  method as d e s c r i b e d e a r l i e r  (4).  Each cuvette  c o n t a i n e d 3 nil of a s o l u t i o n o f c a l f thymus DNA Freehold,  (Worthington,  N.J.) o f s u i t a b l e c o n c e n t r a t i o n i n 0.1M ammonium  a c e t a t e b u f f e r , pH 6 . 8 .  Unless otherwise  s t a t e d , 15 u l o f  enzyme s o l u t i o n , c h i l l e d i n an i c e - b a t h , was added to the cuvette a t zero time. with a p l a s t i c  stirrer.  Mixing was e f f e c t e d by r a p i d l y s t i r r i n g The l i n e a r  change i n absorbance a t  260 mu was f o l l o w e d i n a G i l f o r d 2000 u s i n g b u f f e r s o l u t i o n as a blank.  Spectrophotometer,  Where the c o n c e n t r a t i o n of  s u b s t r a t e used was too high and exceeded the range o f the instrument,  a s u b s t r a t e - b u f f e r blank was used.  was expressed  DNase a c t i v i t y  i n terms o f the i n c r e a s e i n absorbance a t 260  mu per minute (AO.D. per min). The c a l f thymus DNA used i n these to  s t u d i e s was found  c o n t a i n 9 . 2 $ by weight o f phosphorus by the F i s k e and  SubbaRow method ( 8 6 ) .  I n the r e s u l t s r e p o r t e d l a t e r , where  i t a p p l i e s , the c o n c e n t r a t i o n s o f DNA are expressed  i n terms  -71o f DNA phosphorus (DNA-P).  I n the a c t u a l experiments, these  were determined by d i r e c t weighing o f DNA. Where the e f f e c t s o f b i v a l e n t metal i o n s were i n v e s t i g a t e d , these were added as the c h l o r i d e s to the DNA s o l u t i o n to g i v e the d e s i r e d f i n a l c o n c e n t r a t i o n s . crease  i n c a t i o n and DNA c o n c e n t r a t i o n s  The de-  due to the a d d i t i o n  of enzyme was ignored i n the c a l c u l a t i o n s s i n c e t h i s  involved  no more than a 2% i n c r e a s e i n volume even when 50 jii o f enzyme s o l u t i o n were added.  The DNase i n h i b i t o r s rased were  a l s o i n c o r p o r a t e d i n t o the s u b s t r a t e bated with the metals before The  s o l u t i o n s and p r e i n c u -  the a d d i t i o n o f enzyme.  spectrophotometric  method was used because o f  i t s s i m p l i c i t y and because i t was f e l t to give the most accurate  measure o f i n i t i a l v e l o c i t y , an e s s e n t i a l r e q u i r e -  ment i n k i n e t i c s t u d i e s , although one o f t e n honoured more i n breach than i n o b s e r v a t i o n . 2.  Metal B i n d i n g The  Studies  binding of Mg  + +  and M n  ++  i o n s by DNA under the  c o n d i t i o n s used f o r a c t i v i t y measurements was s t u d i e d by conductimetry. The  water used i n c o n d u c t i v i t y experiments was twice  -72-  glass-distilled.  Just p r i o r to use, a f u l l bottle of t h i s  water was boiled for 30 mins and the b o t t l e capped while i t was  s t i l l hot.  water.  This helped to remove CO2 dissolved i n the  Water prepared t h i s way had a conductance of l e s s 1  fi  than 5 x 1 0 ~ ° ohms  . A l l the apparatus were steamed for  15 mins to remove ions adsorbed on the glass surface.  A  piece of apparatus was deemed suitable for use i f the conductance of a sample of water did not increase a f t e r having standing i n i t for 15 to 16 hours.  been l e f t  Conductance measurements were made through the courtesy of Mr. L. Neering, on a conductivity bridge b u i l t by the Department of Chemistry, UBC.  This employed a high  frequency (2000 cycles) o s c i l l a t o r amplifier n u l l detector to obtain the balance point ( 8 7 ) . The temperature of measurement was 25.OOC, maintained by a c i r c u l a t i n g o i l bath. 3.  Optical Rotary Dispersion Studies Calf thymus DNA was dissolved to a concentration of  about 2 . 3 2 6 0 A  u  n  i  t  s  P  e r  ml i n 0.1M ammonium acetate buffer,  pH 6 . 8 , containing 0 . 0 1 moles per l i t e r of magnesium or manganese chloride.  The o p t i c a l rotary dispersion (ORD) was  measured over the range 2 2 0 - 3 0 0 mu by means of a Jasco 5 Spectropolarimeter  against a salt-buffer blank.  To eliminate  any non-specific e f f e c t s that cations might have on the ORD  -73-  spectrum of DNA, as the counter  a c o n t r o l was ion.  used with Na  +  (as the c h l o r i d e )  These s p e c t r o p o l a r i m e t r y  measurements  were k i n d l y c a r r i e d out by Miss Ruth K r e h b i e l . RESULTS AND  DISCUSSION  To e l u c i d a t e enzyme mechanisms by k i n e t i c s t u d i e s , a g e n e r a l procedure i s to set up a r a t e equation p e r t i n e n t e q u i l i b r i a e x i s t i n g i n the system. equation and  i s g e n e r a l l y expressed  forming  This rate  i n terms of f r e e s u b s t r a t e  free a c t i v a t o r concentrations.  ficulty.  from the  T h i s can r e p r e s e n t a d i f -  For example, a metal a c t i v a t o r may  operate  by  an e s s e n t i a l p a r t of the a c t i v e center of the enzyme  as w e l l as by forming  a m e t a l l o s u b s t r a t e which i s the  true  s u b s t r a t e , so t h a t the f r e e c o n c e n t r a t i o n s of these components are v i r t u a l l y unknown.  Rate equations  expressed  in  t o t a l c o n c e n t r a t i o n s of s u b s t r a t e and a c t i v a t o r are a l s o f r e quently reason  too complicated  to be of p r a c t i c a l use.  to b e l i e v e (see l a t e r ) t h a t the i n t e s t i n a l  nuclease  There i s deoxyribo-  I needs both a m e t a l l o s u b s t r a t e and f r e e metal a c t i -  v a t o r to f u n c t i o n p r o p e r l y . when i t was  A further complication  d i s c o v e r e d t h a t s u b s t r a t e DNA  t i o n when present  i n h i b i t s the  i n high c o n c e n t r a t i o n s .  deemed a d v i s a b l e to d e r i v e equations  arose  I t was  reac-  therefore  i n terms of some other  parameter, namely, the c o n c e n t r a t i o n of  metallosubstrate.  -74To d e s c r i b e the formation o f the m e t a l l o s u b s t r a t e complex, I t can be assumed t h a t each b i n d i n g s i t e i s a p a i r o f phosphodiester  bonds, termed (PDNA^2 * =  T  h  e  D  N  molecule  A  i s p o r t r a y e d as a s e r i e s o f independent b i v a l e n t anions; the a s s o c i a t i o n i s viewed as a s i n g l e - s t e p formation o f the complex M - ( P  D N A  )0.  T h i s assumption i s not without  McLean and Hastings  precedent.  (88) d e s c r i b e d the b i n d i n g o f c a l c i u m  i o n s to p r o t e i n i n an analogous manner. I n the case o f DNA, the b i n d i n g i s then M  + +  +  (PDNA>2  v  =  M-(P  A d i s s o c i a t i o n constant  |> K  M.DNA  =  + +  ]  ~Z  D N A  ),  represented  2  can be formulated:  [(PPNA)2 ] =  ;  -^j  L>-(P NA)2] D  where b r a c k e t s i n d i c a t e molar c o n c e n t r a t i o n s . I f the t o t a l c o n c e n t r a t i o n o f DNA phosphorus i s 2p_, the t o t a l c o n c e n t r a t i o n o f the b i v a l e n t l i g a n d i s p_. I f , f u r t h e r , the c o n c e n t r a t i o n s o f the complex i s £, the f r e e DNA l i g a n d c o n c e n t r a t i o n i s b, the t o t a l metal c o n c e n t r a t i o n i s m and the f r e e metal c o n c e n t r a t i o n i s a, the f o l l o w i n g equations  hold: K  M.DNA = S ' P / H  1  -75Substituting  K  M.DNA  or  q. K M . DNA  i.e.,  q  =  (m - £ ) ( £  =  £ -P_a-mq +  / b  -b ± =  <  D  A  be  )  + mp_  gives  £  4  BE  5  =  0  solved with  6  the  quadratic  formula:  4ac  -  z  N  +  1  —————————————————————  Substituting  q = |(m  M  6 can  Equation A  K  3 into  - a.)/  2  + £  - q(m  2  2 and  Equations  + £ + K The  M > D N A  )  final  q for X gives  + k  the r e l a t i o n s h i p  + £ + K  /Tm  M > D N A  ) ^ - 4mp_  7  7 u s e d i n the  form of Equation  calcu-  lation i s :  [M-(P  D N A  )2]  = *(CMj + ItDNA-S t  +  K . N .) ± i/(CMj + *LDNA-PJ M  D  t  A  o where [jDNA-Pjis phosphorus*  and^MJ^ =  All  but  K  M >  o f KJVJ.DNA depends on medium binding  (57).  the  total  E>N  A  the  total  concentration  concentration  In Equation pH  and  Several authors  of n u c l e i c a c i d s with  the  (57,  8 are ionic  58)  who  of metal  known.  of  DNA  ions.  The  strength of  value the  i n v e s t i g a t e d the  m e t a l i o n s have  reported  -76d i f f e r e n t v a l u e s f o r the d i s s o c i a t i o n constants. p l e , Zubay and Doty (58)  r e p o r t e d an a s s o c i a t i o n constant  280, with Mg"" i n a system c o n t a i n i n g 0.2M t  of 3.6  found t h a t a t an i o n i c s t r e n g t h of 0.15 x 10~3f[  (0.1M  i  t  n  present.  and 0.16  of  This corres-  x 10~^M.  Wiberg  (57)  and pH 7. Mg.DNA K  the system used i n the present  ammonium a c e t a t e , pH 6.8),  between 0.1  NaCl.  +  ponds to a d i s s o c i a t i o n constant  8.0  For exam-  =  studies  the i o n i c s t r e n g t h v a r i e d  depending on the amount of metal s a l t s  These c o n d i t i o n s are s i m i l a r to t h a t used by Wiberg.  A rough check by conductance measurements on s o l u t i o n s of i n 0.1M  ammonium a c e t a t e , pH 6.8  DNA  and 0.02M with r e s p e c t to  MgCl£ ( i o n i c s t r e n g t h = 0.16), showed a d i s s o c i a t i o n constant of 7.52  x 10~^M.  However, s i n c e t h i s value was  obtained  a s i n g l e - p o i n t determination,  whereas that of Wiberg's  based on s e v e r a l experimental  p o i n t s o b t a i n e d by two  methods, i t was  used.  E f f e c t of Substrate The  at a M g  ++  M g  D  N  A  S i m i l a r l y , the d i s s o c i a t i o n constant  ganese-DNA complex used was 1.  was  different  f e l t that h i s value would be more a c c u r a t e .  Hence, i n a l l c a l c u l a t i o n s , the value o f K was  from  3.64  (DNA)  x 10""%  o f 8.0x10-3^ of the  man-  (57).  Concentration  e f f e c t o f s u b s t r a t e c o n c e n t r a t i o n was  c o n c e n t r a t i o n of 0.003M.  studied  When the i n i t i a l v e l o -  CONC. Fig.  8  OF  DNA- P  (M x 10 )  Plot of i n i t i a l velocity vs.concentration of DNA i n t h e m a g n e s i u m - a c t i v a t e d i n t e s t i n a l DNase I r e a c t i o n . Cone, o f Kg + = 0 . . 0 0 3 M . +  -78cities  (v, i n A o . D . p e r min) were p l o t t e d a g a i n s t the molar  c o n c e n t r a t i o n o f DNA phosphorus ( F i g . 8 ) , i t i s seen t h a t up to 7 . 5 x lO'^M DNA-P, a normal r e c t a n g u l a r hyperbola observed. velocity  was  At h i g h e r s u b s t r a t e c o n c e n t r a t i o n , the i n i t i a l decreased  u n t i l i t became zero a t a DNA-P concen-  t r a t i o n o f 2 2 . 5 x 10""^M. G r e g o i r e and Gregoire  Working with p a n c r e a t i c DNase I,  ( 8 9 ) observed a s i m i l a r  i n h i b i t i o n by  h i g h s u b s t r a t e c o n c e n t r a t i o n , the i n h i b i t i o n being  particu-  l a r l y pronounced when the magnesium c o n c e n t r a t i o n was low. Cavalieri  and Hatch ( 9 0 ) , u s i n g constant  Mg  + +  concentration  w i t h p a n c r e a t i c DNase I , found t h a t the curve o b t a i n e d "by plotting  the i n i t i a l v e l o c i t i e s a g a i n s t s u b s t r a t e  t i o n passed through a maximum and then d e c l i n e d .  concentraThey  attri-  buted t h e i r f i n d i n g s to i n h i b i t i o n by s u b s t r a t e , although no reason was g i v e n why t h i s should be the case.  However, s i n c e  the i n t e s t i n a l DNase I r e q u i r e s b i v a l e n t metal i o n s f o r a c t i vity  (see below), and the experimental  evidence  ( 5 7 , 58)  p o i n t s to the f a c t t h a t DNA binds these c a t i o n s , i t seems q u i t e p o s s i b l e t h a t the true s u b s t r a t e f o r the enzyme i s a metallosubstrate.  Such a circumstance  would permit  the i n h i -  b i t i o n by DNA s u b s t r a t e to be e x p l a i n e d on the b a s i s o f comp e t i t i o n o f f r e e DNA w i t h the true s u b s t r a t e f o r the b i n d i n g s i t e on the enzyme.  -792.  E f f e c t o f Metal Ion Concentrations The  i n t e s t i n a l DNase I shows an absolute  for  b i v a l e n t metal i o n s .  and  Ni  Co  + +  Four metal i o n s , Mn , Mg++, C o ++  were t e s t e d f o r t h e i r a c t i v a t i n g c a p a c i t y .  were found to be the most e f f e c t i v e .  + +  requirement  Mn  ++  + +  and  F o r example, a t -4  a DNA phosphorus c o n c e n t r a t i o n cation concentration,  Mn  ++  o f 1 . 5 x 10  e f f e c t e d an i n i t i a l i n c r e a s e i n  absorbance o f 0 . 1 5 9 p e r min and C o Mg  M and 0.002M  + +  0 . 1 4 4 p e r min, whereas  produced o n l y an i n c r e a s e o f 0 . 0 5 p e r min and N i  + +  + +  less  than 0 . 0 2 p e r min. I t was observed t h a t the i n t e s t i n a l enzyme  attacks  n a t i v e DNA about 5 times a s f a s t as i t does heat-denatured DNA.  The d i f f e r e n c e i n i n i t i a l r a t e s observed c o u l d be due  to d i f f e r e n c e s i n the secondary s t r u c t u r e o f DNA i n the presence o f v a r i o u s  cations.  However, o p t i c a l r o t a t o r y d i s p e r s i o n  s t u d i e s d i d n o t show any s h i f t o f the whole Cotton E f f e c t i n the  s p e c t r a o f s o l u t i o n s o f DNA c o n t a i n i n g d i f f e r e n t k i n d s o f  ions.  The only d i f f e r e n c e i n ' t h e s e ORD s p e c t r a was a s l i g h t  change i n the magnitude o f the peak and trough. When the e f f e c t s o f v a r y i n g metal were s t u d i e d a t f i x e d c o n c e n t r a t i o n s r e s u l t s were observed. concentration  concentrations  o f DNA, some i n t e r e s t i n g  A p l o t of i n i t i a l v e l o c i t y against  o f Mg++ i s seen i n F i g . 9 .  This i s a r e c t -  -80-  '  1  2  C o n e , of M Fig. 9  3  4  5  ( M X10 ) 3  g  total  P l o t o f i n i t i a l v e l o c i t y o f the i n t e s t i n a l DNase I r e a c t i o n a g a i n s t t o t a l c o n c e n t r a t i o n o f Mg *. Cone, o f DNA-P i n r e a c t i o n mixture = 3xl0-'+M. Volume of enzyme ( P r e p a r a t i o n I) used per cuvette = 15 u l . +  6  -81-  angular hyperbola, resembling the normal substrate concent r a t i o n curve.  A similar curve was obtained when the  reaction rate was p l o t t e d against the concentration of added Ni  + +  .  However, when the concentration-activation function  for Mn ++  Mh^,x  ++  was plotted, a maximum was reached at about  SOxlO'^M h, K  when the concentration of DNA phosphorus was 1 . 5 x 1 0 M  (Fig. 1 0 ) .  In another experiment, the data for which are  shown i n Table IX, a second preparation of enzyme (about one f i f t h ' as potent) was used. not reached u n t i l the Mn  ++  I t was found that the maximum was concentration was 200X10~4M.  Also  i n t h i s same set of experiment (Table IX), i t was observed that asethe concentration of DNA-P was decreased,  the i n h i -  b i t i o n began to appear at lower concentrations of Mn . In++  h i b i t i o n by high Mg  ++  concentration also became evident at  low concentrations of DNA-P (Table X). Mg  ++  The concentration of  at which i n h i b i t i o n occurred also appeared to be a func-  t i o n of the substrate concentration. The i n h i b i t o r y effect of high metal i o n concentration on pancreatic DNase I was reported by Kunitz ( 4 ) .  M&yaji and  Greenstein ( 9 1 ) , Desreux et a l . (51) and several others. Kunitz ( 4 ) interpreted the i n h i b i t i o n as mainly due to an effect of increased i o n i c strength.  But t h i s interpretation  was contradicted ( 5 4 ) by the activating effect of C a  + +  which  was maximum at a much higher i o n i c strength than was optimum  0  2  Fig.  4 6 C o n e , of 10  8 Mn  10 t Q t a |  12 14 ( M X 10 )  16  3  P l o t of i n i t i a l T e l o c i t y o f t h e i n t e s t i n a l DNase I reaction a g a i n s t t o t a l concentration o f Mn"*"l'. Cone, of DNA-P i n reaction m i x t u r e = 1.5 x I O - ^ M .  18  20  -83for Mg  + +  activation.  Also t h i s i n t e r p r e t a t i o n c o u l d not  account f o r the i n h i b i t i o n a t low M g the c o n c e n t r a t i o n o f DNA was low, t i n a l enzyme.  + +  c o n c e n t r a t i o n when  as observed w i t h the i n t e s -  M i y a j i and G r e e n s t e i n  ( 9 1 ) concluded  from  t h e i r data t h a t the optimal a c t i v a t i o n o c c u r r e d when the mole r a t i o o f metal t o n u c l e a t e phosphorus was 3 / 1 .  This i s  c l e a r l y not the case with the i n t e s t i n a l enzyme, f o r which the optimal r a t i o was about 3 0 / 1 with M n and 1 5 0 / 1 (when a l e s s potent  ++  a s shown i n F i g . 10  enzyme p r e p a r a t i o n was used) as  shown l a t e r . T h i s phenomenon o f optimal a c t i v a t i o n can be s a t i s f a c t o r i l y e x p l a i n e d i f i t i s assumed t h a t the true  substrate  i s a m e t a l l o s u b s t r a t e , which binds to the enzyme v i a both the s u b s t r a t e and metal ends o f the complex, and b i n d i n g through the metal end i s c o m p e t i t i v e l y i n h i b i t e d by the presence o f excess  metal.  The argument t h a t the m e t a l l o s u b s t r a t e s u b s t r a t e i s n o t without (53)  prepared  Mg  ++  foundation.  and M n  ++  Erkama and Suutarinen  s a l t s o f DNA and c a r r i e d  s t u d i e s o f p a n c r e a t i c DNase I d i g e s t i o n o f these and without  added M g . ++  observed without addition of Mg . ++  i s the t r u e  parallel  salts  with  H y d r o l y s i s o f the MgDNA s a l t was  added f r e e M g  + +  but the r a t e i n c r e a s e d on  No e x p l a n a t i o n was o f f e r e d f o r the d i f f -  -84erence, but i t i s p o s s i b l e t h a t the a d d i t i o n o f e x t r a M g h e l p e d t o keep the DNA i n the m e t a l l o s u b s t r a t e 3.  F u r t h e r S t u d i e s o f Substrate  ++  form.  Inhibition  The a c t i v i t y - s u b s t r a t e c o n c e n t r a t i o n r e l a t i o n s h i p of the i n t e s t i n a l DNase I was f u r t h e r s t u d i e d a t lower concentration of Mg  ++  than t h a t r e p o r t e d i n F i g . 8 .  When  t h i s was done, u s i n g 1 5 u l o f enzyme p e r cuvette, the r e a c t i o n r a t e became too low to be a c c u r a t e l y determined.  The amount  of enzyme was t h e r e f o r e i n c r e a s e d to 5 0 pi P *" cuvette, and e  four concentrations of Mg 7.5  x l O ^ M and 1 . 8 7 x  ++  were used: 3 0 x lO'^M, 1 5 x lO-^M,  lO'Sl.  o f l / v a g a i n s t l/£ was made.  The double r e c i p r o c a l p l o t  (92)  The p o i n t s o b t a i n e d with a M g  ++  c o n c e n t r a t i o n o f 1 . 8 7 x lO'^M were too s c a t t e r e d to be d e f i nitive.  With M g  ++  c o n c e n t r a t i o n s o f 3 0 and 1 5 x l c V , the  points followed e s s e n t i a l l y a straight l i n e .  Data f o r the  former a r e t a b u l a t e d i n Table VIII and p l o t t e d i n F i g . 1 1 . Also shown i n F i g . 1 1 i s the double r e c i p r o c a l p l o t •. _4 a t a Mg c o n c e n t r a t i o n o f 7 . 5 x 1 0 M. As can be seen, t h i s p l o t shows a d i s t i n c t upward c u r v a t u r e .  There i s the temp-  t a t i o n to argue t h a t i f f r e e DNA i s indeed an i n h i b i t o r , i n c r e a s i n g the t o t a l DNA c o n c e n t r a t i o n i s tantamount to i n c r e a s i n g the i n h i b i t o r c o n c e n t r a t i o n , curved double r e c i p r o c a l p l o t .  thus r e s u l t i n g i n a  The f a l l a c y i n t h i s argument  Table VIII  Concentration of DNA-P M.  Table o f v a l u e s o f v (A O.D. p e r min), £ ( M ) , 1/v (min p e r A O.D.) and 1/q (M~l) a t two c o n c e n t r a t i o n s o f t o t a l Mg+T. F i f t y u l o f enzyme ( P r e p a r a t i o n I ) were used p e r c u v e t t e .  Mg++  = 7 . 5 x 10"4M  1/v  V  Mg  1/<1 xlO-4  xlO  V  ++  = 3 0 x lO-^M  3. xl04  1/v  xlO^  xlO^  96.8  0.25  10.3  4.0  300  0.75  3.3  1.33  40.8  0.13  24.5  7.7  122  0.40  8.2  2.5  17p0  0.065  58.8  15.2  78  0.2  12.8  5.0  0.75 x 10"^  15.2  0.035  65.8  28.6  33  0.125  30.3  8.0  0.375 x 10-^  7.3  0.0165 136  61  10.5  0.049  95.2  20.2  0.25 x 10-4  4.0  0.0125  250  80  11.8  O.O37  85  27  0.187  3.2  0.011  313  90  8.9  0.026  112.4  38.4  6 x IO"  4  3 x 10"^ 1.5 x 10  -4  x 10"^  3  xlO-4  -86-  F l g . 11  P l o t o f 1/v a g a i n s t l/q_ f o r t h e i n t e s t i n a l DNase I r e a c t i o n a t two concent r a t i o n s o f M g . v 30 x • © 7.5 x 10" M. ++  4  -87i s that since free DNA and the metal-DNA complex are i n equilibrium, the ratio of inhibitor/substrate concentration should remain constant, provided that neither the metal nor free DNA  i s i n great excess.  The double r e c i p r o c a l plot  should be l i n e a r , as i s the case with 15 and 30 x lCT^M mg concentration.  ++  No satisfactory explanation can be offered  for t h i s curved double r e c i p r o c a l p l o t .  One p o s s i b i l i t y i s  that, as i s shown l a t e r , while the rate equation the deoxyribonuclease Michaelis-Menten  governing  reaction has the form of the c l a s s i c a l  equation  (93)»  i t also contains, i n both the  numerator and denominator, terms of concentration of free metal ions.  These terms can be regarded as constant  ( a  condition which must be f u l f i l l e d to obtain l i n e a r double r e c i p r o c a l plots) i f the amount of metal ions removed by combination with DNA was concentration.  small compared with the t o t a l metal  This condition does not hold when the t o t a l  metal ion concentration i s low.  Another possible explanation  i s that one enzyme molecule might bind more than one molecule o fffie.t.al*ENAcomplex.>isthus resulting i n a curved LineweaverBurk p l o t . 4.  Determination  of Kinetic Parameters  The experiments consisted of measuring the i n i t i a l v e l o c i t i e s of the reaction at varying amounts of  -88added s u b s t r a t e and metal a c t i v a t o r .  The e x p l o r a t o r y expe-  riments d e s c r i b e d e a r l i e r had y i e l d e d enough i n f o r m a t i o n f o r choosing  p r a c t i c a l ranges o f s u b s t r a t e  (where f i r s t  order  k i n e t i c s hold) and metal i o n c o n c e n t r a t i o n s f o r use. DNA, the range s t u d i e d was between 0 . 3 and 3 x 10 pfopsphorus.  The c o n c e n t r a t i o n o f M n  and 200 x l O ' S l and t h a t o f M g The  ++  ++  For  M DNA  was v a r i e d between 10  between 50 and 2 0 0 x  IO-^M.  c o n c e n t r a t i o n s o f the metal-DNA complex were  computed a s d i s c u s s e d e a r l i e r .  T h i s q u a n t i t y i s r e f e r r e d to  as q i n a l l the t a b l e s and graphs. r e p r e s e n t e d by v .  The i n i t i a l v e l o c i t y i s  These data and c a l c u l a t i o n s , together  with  values o f 1/v, l / q and q/v are summarized i n  the numerical  Tables IX, XI and X I I I f o r manganese and Tables IV and VI for  magnesium. At f i x e d c o n c e n t r a t i o n s o f metal,  i n i t i a l velocity  the maximum  (V^gg) and d i s s o c i a t i o n constant K PP o f the a  E-M-S complex were o b t a i n e d by p l o t t i n g the data i n three ways: 1) the v vs. q p l o t ; 2 ) the Lineweaver-Burk p l o t o f 1/y q  a g a i n s t l / q ( 9 2 ) and 3 ) the Hanes p l o t o f q/v a g a i n s t ( 9 ^ ) . Both the slopes and i n t e r c e p t s were used to d e t e r -  mine the best v a l u e s o f the d e s i r e d parameters. are presented for  magnesium.  These p l o t s  i n F i g s . 12 to 15 f o r manganese and i n F i g . 16 I n some o f the p l o t s , the p o i n t s were f a i r l y  Table IX.  I n i t i a l v e l o c i t i e s o f the i n t e s t i n a l DNase I r e a c t i o n a t v a r y i n g c o n c e n t r a t i o n s o f DNA phosphorus and manganese. Initial velocities were expressed i n & O . D . p e r min. F i f t e e n u l o f enzyme (Prep. I I ) were used i n each c u v e t t e .  Concentration of DNA-P MxlO-+  C o n c e n t r a t i o n o f t o t a l Mn++  M x 1 0  4  hO  2 0  30  50  7 0  1 0 0  3.0  0 . 0 2 5 0  0 . 0 3 5 4  0.0508  0 . 0 6 7 0  0 . 0 7 6 2  0.0800  0 . 0 9 2 0  0.0845  1 . 5  0 . 0 2 3 8  0 . 0 3 1 5  0 . 0 3 9 0  0 . 0 5 8 2  0 . 0 5 9 0  0 . 0 6 0 0  0 . 0 6 7 0  0 . 0 5 4 2  1.2  0.0222  0.0246  0.0364  0.0480  O.O535  0.0537  0.0575  0 . 0 4 5 5  0.9  0 . 0 1 7 5  0.0204  O.O303  0.0400  0 . 0 4 1 3  0.0480  0 . 0 4 8 5  0 . 0 4 5 2  0.6  0.0124  0.0177  0.0260  O.O365  0 . 0 3 9 5  0.0430  0 . 0 3 5 8  0 . 0 3 5 8  0.3  0 . 0 0 6 3  0 . 0 1 0 9  0 . 0 1 2 1  0 . 0 3 1 7  0 . 0 2 9 5  0.0240  0 . 0 2 3 0  O.OI85  1 5 0  2 0 0  Table X.  I n i t i a l v e l o c i t i e s o f the i n t e s t i n a l DNase I r e a c t i o n a t v a r y i n g c o n c e n t r a t i o n s o f DNA phosphorus and magnesium. I n i t i a l v e l o c i t i e s a r e expressed i n & 0 . D . per min. F i f t e e n u l o f enzyme ( P r e p a r a t i o n I I ) were used i n each c u v e t t e .  Concentration of DNA-P M x IO " 24  C o n c e n t r a t i o n o f t o t a l Mg++ 50  75  MxlO^  100  150  200  3.0  0.0111  0.0159  0.0160  0.0204  0.0247  1.5  0.0109  0.0145  0.0149  0.0158  0.0189  1.2  0.0094  0.0128  0.0140  0.0147  O.OI55  0.9  0.0080  0.0114  0.0122  0.0133  0.0068  0.6  0.0064  0.0086  0.0090  0.0085  0.0058  0.3  0.0056  0.0074  0.0078  O.OO55  0.0030  Table X I .  Molar c o n c e n t r a t i o n ( x 10*) o f t h e Mn-DNA complex a t v a r i o u s c o n c e n t r a t i o n s o f DNA and t o t a l manganese a s c a l c u l a t e d according to Equation 8 described i n t e x t .  C o n c e n t r a t i o n o f t o t a l M n + + M x 10^  Concentration of  DNA-P  M x 104  10  20  30  50  70  100  150  200  3.0  0.315  0.525  0.675  0.865  1.00  1.10  1.15  1.25  1.5  0.160  0.265  0.340  0.440  O.53  0.56  0.61  0.63  1.2  0.130  0.210  0.290  O.33  0.40  0.45  0.48  0.50  0.9  0.095  0.16  0.20  0.21  0.29  0.33  0.36  0.38  0.6  0.06  0.11  0.15  0.175  0.19  0.20  0.25  0.26  0.3  0.03  0.05  0.07  0.09  0.10  0.11  0.12  0.13  Table X I I .  Molar c o n c e n t r a t i o n ( x 10*) o f the Mg-DNA complex a t v a r i o u s c o n c e n t r a t i o n s o f DNA and magnesium as c a l c u l a t e d a c c o r d i n g to Equation 8 as d e s c r i b e d i n t e x t .  Concentration of DNA-P M x 10^  Concentration o f t o t a l M g  ++  MxlO^ 200  50  75  100  3.0  o.6o  0.71  0.75  0.97  1.0  1.5  0.275  0.36  0.40  0.44  0.575  1.2  0.25  0.29  0.32  0.38  0.45  0.9  0.175  0.215  0.275  0.290  0.325  0.6  0.1  0.145  0.16  0.195  0.22  0.3  0.075  0.092  0.125  0.1317 0.135  150  Table X I I I .  LDNA-P]  MxlO^ 3.0  Ratio  1.2  Goncentration of t o t a l M n  + +  M x 10^  20  30  50  70  100  150  40.5 3.17 12.75  28.2 1.9 14.8  19.7 1.48 13.3  14.9 1.17 10  13.1 1.0 13.1  12.5 0.91 13.75  10.9 0.87 12.55  11.8 0.8 14.75  i/a  42.7 6.25 6.82  31.7 3.77 8.4  25.7 2.94 9.3  17.4 2.27 7.66  16.9 1.89 8.96  16.7 1.79 9.35  14.9 1.64 9.09  18.5 I . 59 I I . 65  a/Z 1/v i/a a/z  45.1 7.69 5.85  40.6 4.76 8.5  27.5 3.45 7.9  20.9 3.03 6.88  18.7 2.5 7.48  18.6 2.22 8.37  17.4 2.08 8.35  22 2.0 11  57.2 10.53  5M  49.1 6.25 7.86  32 5.0 6.25  25 4.76 5.25  24.2 3.^5 5.06  20.9 3.0 6.9  20.6 2.78 7.42  22.2 2.63 8.44  80.6 16.66 4.83  56.5 9.0 6.21  38.5 6.7 5.75  27.4 5.71 4.79  25.4 5.26 4.83  23.3 5.0 4.66  28.0 4.0 7.0  28.0 3.85 7.28  158 33*3 4.74  91.7 20 4.59  82.6 14.3 5.8  31.5 11.36 2.77  33.9 10 3.39  41.7 9.1 4.58  43.5 8.33 5.22  54.1 7.69 7.03  1/v  i/a  1/v 0.9  x  10  1/v 1.5  Table o f v a l u e s o f 1/v ( min p e r A O.D.); 1/a, (M*" x 10~*) and q/v ( M. m i n / A o . D . , x 10 ) o f t h e manganese-activated DNase I reaction.  1/1  a/z 1/v  0.6  1/5 a/z  0.3  i/a a/z  1/v  200  Km  0  Cone, Fig.  12  0.1 of  0.3  0.2 Mn-( DNA^2 P  M X 10  4  P l o t o f i n i t i a l v e l o c i t y a g a i n s t cone, o f Fin-DNA complex f o r ' the Mn' - a c t i v a t e d i n t e s t i n a l DNase I r e a c tiso n . = • 10 x 10" C o n c e n t r a t i o n o f t o t a l Mn  -1 Fig. 14  M~  q  1  X 10~  4  P l o t o f 1 / v a g a i n s t 1/q f o r t h e M n - a c t i v a t e d i n t e s t i n a l DNase I r e a c t i o n a t v a r i o u s c o n c e n t r a t i o n s o f M n . • 3 0 X 1 0 - 4 M I O 50xlO- M; A 7 0 X 1 0 - ^ ; • l O O x l Q - ^ M ; + +  ++  /+  ©150xi0-4 .. M  0  200X10~4M.  14 12  r  o X »  -0  I  cr >  F i g . 15  q  M. X 10  P l o t o f £/v a g a i n s t a f o r t h e M n - a c t i v a t e d i n t e s t i n a l DNase I r e a c t i o n a t v a r i o u s c o n c e n t r a t i o n s o f M n . © lOxlOT^M; * 20x10-^1; a30xlO-^M; O50xl0-%j; A 7 0 X 1 0 - ^ M ; lOOxlO-^M; © 1 5 0 X 1 0 - ^ 1 ; E 200xlO-Vi. + +  ++  -99-  scattered.  F o r t h e sake o f c l a r i t y , o n l y t h e p o i n t s which  r e p r e s e n t t h e g i v e n l i n e a r e i n c l u d e d i n t h e graphs.  Nume-  r i c a l d a t a f o r t h e o t h e r p o i n t s may be o b t a i n e d from t h e appropriate tables.  The s c a t t e r i n g o f some o f t h e p o i n t s  may be a t t r i b u t e d t o any o r a l l o f t h r e e a.  causes:  Experimental e r r o r i n determining the i n i t i a l  velocities.  T h i s was e s p e c i a l l y a c u t e when t h e i n i t i a l r a t e s  were l o w . However, such e r r o r s were e l i m i n a t e d t o t h e b e s t e x t e n t by c a r r y i n g o u t t r i p l i c a t e runs i n t h e e x p e r i m e n t a l work. b.  E x a g g e r a t i o n o f metal-DNA b i n d i n g .  There has  been c o n s i d e r a b l e c o n t r o v e r s y c o n c e r n i n g t h e e x t e n t o f b i n d i n g o f m e t a l by DNA.  Zubay and Doty ( 5 8 ) , f o r i n s t a n c e , c l a i m e d  t h a t one n u c l e o t i d e r e s i d u e i n DNA can b i n d a maximum o f 0 . 2 1 ion  of Mg . ++  Shack e t  al.(95)  and Wiberg  (57)  found t h a t  b i v a l e n t m e t a l s combined w i t h DNA i n a maximum r a t i o o f one m e t a l atom p e r two phosphorus atoms.  Before t h i s controversy  i s r e s o l v e d , t h e p o s s i b i l i t y remains t h a t t h e v a l u e s o b t a i n e d for  q t l° &  w  DNA and h i g h m e t a l c o n c e n t r a t i o n s might be  exaggerated. c.  The range o f m e t a l i o n s u s e d was d e l i b e r a t e l y  chosen so t h a t I n h i b i t i o n by h i g h metal c o n c e n t r a t i o n c o u l d be observed.  At these c o n c e n t r a t i o n s o f m e t a l i o n s , t h e  k i n e t i c s o f t h e r e a c t i o n may n o t f o l l o w t h e p a t t e r n s t i p u l a t e d by t h e r a t e e q u a t i o n t o be p r e s e n t e d l a t e r .  The case  observed  -100a t 200X10~4M M n  serves w e l l to I l l u s t r a t e t h i s p o i n t .  + +  This concentration of M n t i o n s o f 0.3 and 0.0  i s i n h i b i t o r y a t DNA-P c o n c e n t r a -4  + +  x 10  M.  The i n i t i a l v e l o c i t i e s a t  these two c o n c e n t r a t i o n s o f DNA t h e r e f o r e depart from t h a t based on s t r i c t Michael!s-Menten k i n e t i c s , a s can be seen from F i g . 14. At h i g h e r c o n c e n t r a t i o n s o f DNA, t h e i n h i b i t o r y power o f M n  + +  i s l e s s severe.  The k i n e t i c b e h a v i o u r o f  the r e a c t i o n appeared more normal ( F i g . 1 4 ) . Even then, a s can be seen l a t e r , t h e v a l u e o f K  a p p  obtained i s unduly l a r g e ,  i n d i c a t i n g that the i n h i b i t o r y e f f e c t of Mn  + +  has caused a  d e c r e a s e i n a f f i n i t y o f t h e enzyme f o r t h e m e t a l l o s u b s t r a t e . 5.  D e r i v a t i o n o f Rate  Equation  The f o l l o w i n g assumptions were made i n d e r i v i n g t h e rate equation f o r the i n t e s t i n a l deoxyribonuclease  reaction:  a. The t r u e s u b s t r a t e f o r t h e enzyme i s a m e t a l l o substrate . b. The enzyme s u r f a c e has a t l e a s t two b i n d i n g one f o r b i n d i n g w i t h m e t a l , t h e o t h e r w i t h DNA.  sites,  The former  s i t e s e r v e s t o anchor t h e m e t a l end o f t h e m e t a l l o s u b s t r a t e complex, t h e l a t t e r s i t e i s a l s o capable  of binding with the  s u b s t r a t e o f t h e complex. c. The f o r m a t i o n o f t h e t e r n a r y enzyme-metal-DNA complex i s by random a d d i t i o n , a l t h o u g h w h i c h e v e r component  -101-  i s added onto the enzyme s u r f a c e f i r s t does not a f f e c t form o f the f i n a l e q u a t i o n .  the  For the sake o f s i m p l i c i t y ,  the  mechanism o f f o r m a t i o n of the t e r n a r y complex by a d d i t i o n o f MS,  the metal-DNA complex, to enzyme i s assumed. The  f o u r e q u i l i b r i a must be  considered: Dissociation  I.  E + M  ^  II.  E + S  u  III.  M + S  S  E + MS  *  IV.  The 1.  wK  2.  rK  3.  4.  p_K  s  =  s  =  AS  EM  K  A  "  ES  K  s  ^  MS  K  0  EMS  K  corresponding =a(  A  .  ^  mass-action equations  e - w - p - r ) s ( e - w - p _ - r )  KQ.II  /  a  = £ ( e - w - £ - r ) In addition,  5.  v = kp_  where  w = c o n c e n t r a t i o n of  EM  a = c o n c e n t r a t i o n of f r e e m e t a l i o n e = t o t a l c o n c e n t r a t i o n o f enzyme p_ = c o n c e n t r a t i o n o f  EMS  q = c o n c e n t r a t i o n of  MS  constant  A S  are:  -102-  r = c o n c e n t r a t i o n o f ES s = c o n c e n t r a t i o n o f t o t a l DNA-P k = r a t e c o n s t a n t o f t h e breakdown o f the EMS complex From e q u a t i o n 1, we have wK = ae - aw - ap - a r A  w( K  + a) = a( e - £ - r ) ,  A  hence  a( e - p_ - r ) w  =  6.  K + a S u b s t i t u t i n g Equation 6 into Equation 2 gives a( e - £ - r ) r K a = s (e - p - r ) K + a ~ A  A  s ( eK  + ae - ae + ap + a r - £ K  A  KA  s ( eKA K  A  £KA  A  - ap - r K - a r ) A  + a  - rKA)  + a  whence rK ( s  K  A  + a) = s K ( e - £ ) - r s K A  A  s K ( e - £) A  K K g + aKg + s K A  A  S u b s t i t u t i n g Equation 7 into Equation 6 gives  -103-  s K ( e - £__) A  a (e - p_  K K A  w =  K aeKjK il  )  + aKg + s K  S  A  + a  A  + aaeKg - apK^Kg - aapKg  g  8.  =  (K  + a)( K K  A  A  + aKg + s K )  S  A  S u b s t i t u t i n g E q u a t i o n s ? and 8 i n t o E q u a t i o n 4 g i v e s a e K K g - aaeKg-apjtftKg-aapKq  £sK  A  P. AS= K  A  - psK  A  p)  :  ( K eK K K A  = q  A  + a ) ( K K g + aKg + s K )  A  s  A  + aeK K A  K K + aKg+_sK  A  A  S  A  - £ K K K g -ap_K Kg  s  A  A  A  (  )  ( K + a )( K K A  A  S  + aKg + s K ) A  Hence,  £ K  AS  ( K  A  +  ^  ) ( K  A S K  +  - S K  +  -V  5l A S - A  =  K  K  (  K  —  +  " £ A K  or aqK K K A  P  " < A A S AS + K  K  K  aaK K s  K  A S  a K  A S AS K  K  +  A  s  + aeqK K A  * A A AS K  K  K  +  s  § A S AS+ K  K  K  + a s K K g + aqK Kg + £K K Kg ) A  A  A  A  A  S u b s t i t u t i n g t h e v a l u e o f s from E q u a t i o n 3 i n t o E q u a t i o n 9, we have  '  -104— A A S K  K  K  P=  +  l A S  a e (  K  0K K K A  (K K K K A  A  S  + aK K K  A S  s  s  A  K  A S  K  0  +  A S  + aK KgK A  A S  +  cX  ^ S AS K  K  ^  +  K A  A S 0 + a£K K K  A  aeqK K K A  ( aK K K K A  A  s  A  + aaK K K  A S  A  aaaKgK g+ a q K K A  A  A g  K  s  Q  g  A S  s  qK^K^K^  +  + aaeqK K A  )  s  + o ^ K ^  + aaK KgK g + A  + aaqK^Kg + a q K K K A  A  A  )  g  a e q K K K g + aaeqK Kg A  A  A  <S ^ A s M a + K  A  ) + agK K (  aK KgK ( a + K  A  ) + aaKgK ( a + K )  A  A g  S A AS 0 K  K  K  B ¥ s  +  A  a + K ) + A  s  AS  * A S AS  +  K  K  K  +  A  a a K  S AS  +  ^ S £S  +  ^ S AS  )  K  v = kp aeqkK Kg A  £ A AS 0 K  K  K  +  ^ A S K  K  +  ^ A S AS K  K  K  K  K  vaqK Kg A  S A AS 0 K  K  K  +  ^ A S K  K  +  2 A S AS K  K  K  K  K  where V = ek, i . e . , t h e v e l o c i t y a t which a l l t h e enzymes a r e i n t h e form EMS.  -105-  \  Equation  11 can be w r i t t e n i n t h e form aK =_5 aK + K K — aK K (a + K ) K ( aK + K K ) s  V  s  v =  Q  s  -  A S  A S  A  .q  12.  A  3  0  A S  At any f i x e d metal i o n c o n c e n t r a t i o n , t h e V^PP and K  a p p  o b t a i n e d a r e r e l a t e d t o t h e i n i t i a l v e l o c i t y as i n t h e  following  equation: q v  v =  max • it  (93)  a + K P a p  Hence, V PP max  =  app  _  a  K  v  ,? aKg + 0 A S aK  K  a K  8 AS ( g + K  )  K  K  A  A  (-aK + K K s  13  K  0  A S  )  From E q u a t i o n 13, i t can be seen t h a t  K  app  1 , 1 V V  KpK s Ks A  l a  A p l o t o f 1/ vf;pp a g a i n s t 1/a s h o u l d g i v e a s t r a i g h t l i n e , and a p l o t o f V ^ P a g a i n s t a s h o u l d g i v e a r e c t a n g u l a r hyperbola.  -106Equation l 4 i s not e a s i l y amenable to treatment. But i f i t i s assumed that a^> K , Equation 14 becomes A  K P = a p  aK K + K K K 2  A  a  =  K PP a  s  K  s  K  2  K  a ~ K  A  S  A  0  K K  A  A  K  A S  A S  0  16,  S  P l o t t i n g a / K P P a g a i n s t a should g i v e a s t r a i g h t 2  line.  a  On the o t h e r hand, i f K .§> a, which i s more reasonable, A  c o n s i d e r i n g the u s u a l s l u g g i s h n e s s with which p r o t e i n s b i n d metal IJionass, Equation 14 becomes  £ £ A  K  S  K  A S  K PP =  17.  a  aK K A  or  1/K  a p p  = 1/K  AS  s  + K K K A  Q  + K /aK Q  A S  18.  s  A p l o t o f l / K P P a g a i n s t l / a should g i v e a s t r a i g h t a  line. The v a l u e s o f Vggg, K PP o b t a i n e d from F i g s . 12 a  through 16 t o g e t h e r with the v a l u e s o f l / W P , l / K P P , 1/a a  and a / K P P a r e summarized i n Tables XIV and XV. 2  a  The v a r i o u s  p l o t s f o r Equations 13, 14, 15, 16 and 18 a r e shown i n F i g s . 17 through 24.  Table X I V  Summary o f k i n e t i c parameters o f t h e i n t e s t i n a l DNase I r e a c t i o n . Manganese was t h e a c t i v a t i n g i o n .  C o n c e n t r a t i o n o f t o t a l Mn"*"", 1  Parameter  —  yaPP Ao.D./min 2  xlO^ app/yapp M/Ao.D., p e r min K  M x10'  1 0  2 0  3 0  5 0  7 0  1 0 0  1 5 0  2 0 0  2 . 5 5  4 . 3 5  6 . 1  8 . 5  8 . 9  1 1 . 1  1 5 . 6  2 0 . 5  3 . 6  3 . 2 8  2 . 8 8  3 . 3 5  3 . 5  5 - 5 5  8 . 2 5  0 . 6  1 . 5 7  2 . 0  2 . 4 5  2 . 9 8  3 . 1 8  8 . 3 3  1 6 . 8  1 0 0 0  5 0 0  3 3 3  2 0 0  1  1 0 0  6 6 . 7  5 0  3 9 . 1  2 3  1 6 . 4  1 2 . 7  1 1 . 2  9 . 0  6 . 4  4 . 9  1 6 . 7  6 . 3 7  5 . 0  4  3 . 3 8  3 . 1 5  1 . 2  0 . 5 6  0 . 1 6 7  0 . 2 5 2  0 . 4 5  1 . 0 2  1 . 6 4  3 . 1 4  2 . 7  -  •xlO*  K PP M x 10 5 a  1/a 4  3  l/V PP a  l/A°« «P D  e r  min  l/K PP a  M-l x  .  0  8  1 0 - ^  a /K PP 2  a  M.  2 . 3 8  Table XV  Summary o f k i n e t i c parameters o f t h e Mg i n t e s t i n a l DNase I r e a c t i o n .  +  Concentration of t o t a l Mg , ++  Parameter V PP A O . D . / min  -activated  M x 10*  5Q  70  100  150  200  1.31  1.96  2.38  2.86  ll.l  1.04  1.52  2.09  3.0  25.9  200  133  100  67  50  76  51  42  35  9.0  9.5  6.6  4.8  3.3  0.39  2.4  3.6  4.8  7.4  1.55  a  xlO  K PP  2  a  M x 105 1/a  l/^PP . / 2 \ 0 . D . p e r min  l/K PP M~l x IO"* a  a /K PP 2  a  M.  F i g . 17  P l o t o f V -°P and K P a g a i n s t c o n c e n t r a t i o n o f f r e e M n the i n t e s t i n a l DNase I r e a c t i o n , n V P P ; • K P P . a  a  p  a  a  + +  for  -110-  F i g . 18  P l o t - o f l / V P P a g a i n s t .1/a f o r the M n - a c t i v a t e d i n t e s t i n a l DNase I reaction. a  + +  -111-  [freeMn] F i g . 19  M  P l o t o f l / K ? P a g a i n s t 1/a f o r the ' M n - a c t i v a t e d i n t e s t i n a l DNase I r e a c t i o n . a  ++  Cone, Fig,  20  of  free  Mn.  M . X 10  P l o t of a /K ?P against concentration of free Mn f o r t h e M n - a c t i v a t e d i n t e s t i n a l DNase I r e a c t i o n . 2  a  + +  + +  F i g . 21  P l o t o f V ^ P and K '°P a g a i n s t c o n c e n t r a t i o n o f f r e e Mg** f o r t h e I % " - a c t i v a t e d i n t e s t i n a l DNase I r e a c t i o n . • V P P ; © K P . a  +  h  a  a p  10  8  K app. M7 xi0~ 1  4  i  4\—  M H  120  160  M -1 F i g . 23  P l o t o f 1/K ^ a g a i n s t 1/a app  i n t e s t i n a l DNase I  f o r the  reaction.  Mg -activated + +  200  I  50  •" Cone,  F i g . 24  of  1  1  I  100  150  200  free  Plot of a / K 2  Mg  + +  Mg.  M. X 1 0  4  against concentration of free  a p p  f o r t h e M g * - a c t i v a t e d i n t e s t i n a l DNase. I  reaction.  +  -117-  I t may be n o t e d t h a t i n these f i g u r e s , t h e concen-' t r a t i o n o f f r e e m e t a l i o n s i s t a k e n t o be t h e same a s t h a t of t h e t o t a l m e t a l i o n s .  The r e a s o n i s t h a t t h e c o n c e n t r a -  t i o n o f DNA used i n these  s t u d i e s i s s m a l l compared t o t h e  c o n c e n t r a t i o n o f m e t a l i o n s , so t h a t even i n t h e extreme case r e p r e s e n t e d by a M n  concentration of  ++  IOXIO'^M  d i e d ) and DNA-P c o n c e n t r a t i o n o f JxlO'^E l e s s t h a n 5% (Table X I ) o f t h e t o t a l M n  (lowest  stu-  (highest studied), was removed..  + +  Thus,  the c o n c e n t r a t i o n o f f r e e m e t a l i o n may be c o n s i d e r e d t o be e s s e n t i a l l y t h e same a s t h e t o t a l m e t a l c o n c e n t r a t i o n . F i g . 1 7 , i n which V  3-1  ^ and K  a p p  are p l o t t e d against  the c o n c e n t r a t i o n o f f r e e M n , shows t h a t up t o a M n ++  e n t r a t i o n o f yOxlO'^K,  the  curve i s a r e c t a n g u l a r hyper-  b o l a , a s p r e d i c t e d by E q u a t i o n 1 3 . Up t p concentration, the K hyperbola,  A  P  P  conc-  + +  Mn  IOOXICT^M  + +  vs. a plot i s also a rectangular  which s h o u l d be t h e case i f E q u a t i o n 17 h o l d s .  higher concentrations o f metal i o n s , the values o f  K  A  P  P  At  rise  r a p i d l y , i n d i c a t i n g that high metal concentration i n t e r f e r e s w i t h t h e combination The  o f t h e metal-DNA complex w i t h t h e enzyme.  r i s e i n value of V  70x10  a p p  at Mn  + +  concentration greater than  M i s n o t so e a s i l y e x p l a i n e d .  I t i s d i f f i c u l t to  v i s u a l i z e how h i g h m e t a l c o n c e n t r a t i o n can f a c i l i t a t e t h e breakdown o f t h e EMS complex. f o r t h i s phenomenon.  No e x p l a n a t i o n can be o f f e r e d  -118-  The  p l o t s o f l / V P P and l / K P P a g a i n s t l / a ( F i g s . a  a  18 and 19) f o r M n  + +  are s t r a i g h t l i n e s i f the points corres-  ponding t o h i g h m e t a l c o n c e n t r a t i o n s a r e i g n o r e d .  As dis-s  cussed b e f o r e , under these c o n d i t i o n s , t h e i n h i b i t o r y e f f e c t s o f t h e m e t a l s supercede t h e e q u i l i b r i a from which t h e r a t e e q u a t i o n was d e r i v e d .  Where t h i s i n h i b i t o r y e f f e c t was  a b s e n t , t h e v a l i d i t y o f t h e r a t e e q u a t i o n was v e r i f i e d by the c o l i n e a r i t y o f t h e p o i n t s . F i g . 20 shows t h a t t h e r e l a t i o n s h i p between a^/K PP a  and a i s n o n - l i n e a r , i n d i c a t i n g t h a t t h e assumption K §>a A  I s indeed  valid. The  p l o t s obtained with Mg  + +  ( F i g s . 21 t o 2k) a r e  s i m i l a r to that f o r Mn , with s i m i l a r reservations f o r i n h i ++  b i t i o n by h i g h M g rate equation  + +  concentration.  i s v a l i d for Mg  + +  This i n d i c a t e s that the  as w e l l as f o r Mn , w i t h i n  c e r t a i n l i m i t s o f metal i o n concentration.  ++  Mg  + +  thus pro-  b a b l y a c t i v a t e d t h e i n t e s t i n a l . D N a s e I r e a c t i o n i n t h e same manner a s M n . ++  Some o f t h e d i s s o c i a t i o n c o n s t a n t s from t h e g r a p h s .  As can be seen from E q u a t i o n  = 0 , l / v P P = 1/V. a  c a n be e v a l u a t e d 1 5 , when 1/a  F o r Mg++, t h e maximum v e l o c i t y a t t a i n a b l e  when a l l t h e added enzyme was i n t h e form o f EMS i s 0 . 1 2 5 Ao.D.  p e r min.  F o r M n , t h e maximum v e l o c i t y a t t a i n a b l e i s ++  -119-  Ao.D.  0.167  p e r min.  at 200x10-*% M n  V ^ 8  However, Table XIV shows t h a t the + +  i s 0 . 2 0 5 Ao.D.  p e r min.  T h i s anomaly  has been mentioned. From E q u a t i o n 18, i t can be seen t h a t when l / a = 0 , l / K P P = 1 / K , and £h'§ s l o p e o f the l / K P P a  a  AS  K /K . 0  v s . 1/a  a x i s i n F i g . 19  For M n , the i n t e r c e p t on the l / K P P ++  s  a  i s 2 . 3 x 1 0 ^ M" .  Thus the v a l u e o f K  1  Since KQ,  A S  plot i s  for Mn  + +  i s 4.35X10"5M.  fHS d i s s o c i a t i o n c o n s t a n t o f the Mn-DNA complex i s  —4  36.4x10  M (57).  the v a l u e o f Kg computed from the s l o p e o f  the p l o t i n F i g . 19 i s 4 . 5 x l 0 ^ M .  Thus, i t can be seen t h a t  _  the enzyme b i n d s f r e e DNA  and the Mn-DNA complex w i t h e q u a l  affinity. S i m i l a r l y , from F i g . 2 3 , of  the E-Mg-DNA complex was  the d i s s o c i a t i o n c o n s t a n t  c a l c u l a t e d t o be  approximately  3 x l 0 ^ M , i n d i c a t i n g t h a t the Mg-DNA complex b i n d s the -  intes-  t i n a l DNase I w i t h much s m a l l e r a f f i n i t y t h a n the Mn-DNA complex.  T h i s a g r e e s w e l l w i t h the f a c t t h a t M n  + +  i s a much  b e t t e r a c t i v a t o r o f the i n t e s t i n a l DNase I t h a n M g . ++  d o x i c a l l y , i t a l s o e x p l a i n s why of  the r e a c t i o n t h a n M g  high.  For, i f K g A  + +  Mn  + +  Para-  i s a better inhibitor  when the m e t a l c o n c e n t r a t i o n i s  r e f l e c t s i n p a r t the a f f i n i t y o f the  en-  zyme f o r the m e t a l , the m e t a l i o n w i t h g r e a t e r a f f i n i t y f o r the enzyme would be a b e t t e r i n h i b i t o r .  -120-  The evidence from t h e s e k i n e t i c s t u d i e s a g r e e s w e l l w i t h t h e mechanism p o s t u l a t e d f o r t h e m e t a l i o n a c t i v a t i o n o f the i n t e s t i n a l DNase, namely, t h e metal-DNA complex i s the t r u e s u b s t r a t e , and b o t h t h e m e t a l and DNA ends a r e i n comb i n a t i o n w i t h the enzyme t o form a t e r n a r y complex.  However,  a t t h i s stage, o t h e r mechanisms have n o t been r u l e d o u t . These a r e c o n s i d e r e d as f o l l o w s : Case 1.  I f the t r u e enzyme s u b s t r a t e i s DNA and t h e m e t a l  a c t s by combining w i t h t h e enzyme i n d e p e n d e n t l y  o f the subs-  t r a t e , the f o l l o w i n g r e a c t i o n s must be c o n s i d e r e d : • E  +  M \  ^ -EM  E  +  S  ^ ES  EM  +  S  ES  +  M ,  EMS  %  EMS  EMS  >  products  U s i n g the same n o t a t i o n s as p r e v i o u s l y , the i n i t i a l velocity  i s d e s c r i b e d by a r a t e e q u a t i o n d e r i v e d from a con-  s i d e r a t i o n o f the above e q u i l i b r i a : aK v  =  AaK  0  Q  + K K A  aK  £ + K  Therefore,  V. g 0  0  -121  v*PP  0  aK K PP a  aK _ + K K  = 0  A  = aK /K s  and 0  Q  I t i s seen t h a t a p l o t o f K line.  a p p  vs. a i s a straight  T h i s was n o t o b s e r v e d ( F i g . 17)  Case 2.  A g a i n , i t i s assumed t h a t m e t a l a c t s by b i n d i n g w i t h  enzyme, and o n l y t h e EM complex can combine w i t h t h e s u b s t r a t e , When t h i s mechanism o f a c t i o n p r e v a i l s , t h e r a t e d e s c r i b i n g i t i s extremely  complicated.  equation  However, such a mech-  anism may be r u l e d o u t on t h e f o l l o w i n g grounds: m e t a l - p r o t e i n i n t e r a c t i o n s a r e u s u a l l y v e r y slow (96), r e q u i r i n g sometimes a p r e i n c u b a t i o n o f t h e m e t a l w i t h t h e p r o t e i n f o r up t o sever a l hours.  I n t h e DNase r e a c t i o n , d e p o l y m e r i z a t i o n o f DNA  as shown by an i n c r e a s e i n absorbance o f t h e DNA s o l u t i o n was o b s e r v e d almost immediately Case 3.  a f t e r t h e a d d i t i o n o f enzyme.  I f t h e t r u e s u b s t r a t e i s t h e metal-DNA complex, b u t  the enzyme does n o t combine w i t h t h e f r e e s u b s t r a t e o r m e t a l , the e q u i l i b r i a t o be c o n s i d e r e d a r e : M  +  S  ,  E  +  MS  ,  —1  MS EMS  >  The r a t e e q u a t i o n d e r i v e d from these a r e v  =  Vq / q  + K  A S  products  -122-  Thus, n e i t h e r V -^ n o r K 3  concentration o f free metals. experimental Case 4.  a p p  a r e a f f e c t e d by t h e  This i s not i n accord  with  observation.  I f , i n a d d i t i o n t o t h e case d e p i c t e d i n Case 3, t h e  enzyme.can combine w i t h f r e e DNA (thus i m p l y i n g by f r e e s u b s t r a t e ) , these e q u i l i b r i a e x i s t M  +  s  E  +  S  E  +  MS  The  „ —  »  MS  *  ES  »  EMS  competition  i n t h e system:  >  products  r a t e e q u a t i o n i n terms o f t h e metal-DNA complex  can be shown t o be  aK  s  + K K 0  a K  A S  S AS K  S. + aK Both t h e l/V -^ 3  s  + K K Q  A S  and l / K  a p p  v s . 1/a p l o t s would be  s t r a i g h t l i n e s , a s found i n these experiments.  However,  such a mechanism negates t h e p o s s i b i l i t y o f i n h i b i t i o n by h i g h m e t a l c o n c e n t r a t i o n , i . e . , t h e EMS complex i s o f t h e form E-S-M.  T h i s c o n t r a d i c t s t h e evidence and t h e mechanism  i s ruled out. Case 5.  I n t h i s case, i t i s assumed t h a t MS i s t h e t r u e  -123s u b s t r a t e , but the l i n k a g e between MS and E i s v i a the metal, and  S does not combine with E.  Implicit  i n t h i s assumption  i s that there i s no competition by f r e e s u b s t r a t e ,  again  c o n t r a d i c t i n g the evidence with the i n t e s t i n a l DNase ( F i g . 8 ) . Kinetically,  i t can be shown t h a t  v =  S AS K  £+ K  Thus experimental  A S  +  i s independent o f a, which i s c o n t r a r y to evidence.  The k i n e t i c  evidence p o i n t s to the c o n c l u s i o n t h a t  i n the case o f the i n t e s t i n a l DNase, the enzyme, DNA and the .M • metal form a t e r n a r y complex o f the form E C • . o  A t e r n a r y complex formed by enzyme, s u b s t r a t e and c o f a c t o r has been p o s t u l a t e d as an i n t e r m e d i a t e  f o r the  enzyme pyruvate kinase by Mildfean and Cohn (97) on the b a s i s of k i n e t i c and magnetic resonance data.  Although the i n t e r -  p r e t a t i o n o f the k i n e t i c  et a l . has been  questioned  data o f Mildvan  by C l e l a n d ( 9 8 ) , t h e i r pre ton.: r e l a x a t i o n 1  suggest the e x i s t e n c e o f a t e r n a r y 6.  Inhibition  data  cpmplex.  Studies  P a n c r e a t i c DNase I has been shown to be i n h i b i t e d by  -124c i t r a t e , a r s e n a t e and EDTA (54,  49,  99,al00).  Similar  f i n d i n g s were r e p o r t e d f o r the i n t e s t i n a l DNase I (46).  The  e f f e c t o f v a r y i n g the c o n c e n t r a t i o n o f these i n h i b i t o r s studied.  The  was 25.  r e s u l t s o f these s t u d i e s a r e shown i n F i g .  As can be seen, EDTA i s the most e f f e c t i v e i n h i b i t o r , f o l l o w e d by c i t r a t e and a r s e n a t e . constant  T a k i n g the d i s s o c i a t i o n  o f the M g - c i t r a t e complex t o be 5.6,  ± 2.1  x  10~^M  (101), i t can be seen t h a t the i n h i b i t i o n by c i t r a t e i s by the removal of magnesium from the r e a c t i o n m i x t u r e . under the c o n d i t i o n s o f study, a 5°$  i n h i b i t i o n was  a t a c i t r a t e c o n c e n t r a t i o n o f 1.5x10""%. of free Mg  ++  all  (over 90$)  rate.  The  At 100$  observed  concentration  a t t h i s l e v e l o f c i t r a t e i s about 60$  t o t a l magnesium added.  Thus,  of  the  i n h i b i t i o n by c i t r a t e ,  o f the added magnesium was  complexed w i t h  Thus i t seems q u i t e c e r t a i n t h a t c i t r a t e i s not  i n h i b i t o r o f the DNase I r e a c t i o n per se. constant  nearly cit-  an  The d i s s o c i a t i o n  of the Mg-arsenate complex was not a v a i l a b l e from  literature.  However, i t seems q u i t e l i k e l y t h a t , l i k e  a r s e n a t e i n h i b i t s the i n t e s t i n a l DNase I r e a c t i o n by  citrate,  binding  magnesium. EDTA, on the o t h e r hand, appears to a c t i n q u i t e a d i f f e r e n t manner.  As can be seen from F i g . 25,  c o n c e n t r a t i o n o f 0 . 8 x 1 0 " % i n h i b i t s o v e r 90$  EDTA a t a  o f the DNase I  1 0.5  1  2.5 M. x 1 0  1.5 C o n e , of Inhibitor  25  1  3  Plot of i n i t i a l velocity against concentration o.f i n h i b i t o r s f o r t h e i n t e s t i n a l DNase I r e a c t i o n . ^4, Cone, o f DNA-P = 3xl0-" M. Cone, o f Fig =3xl0-^M. I n h i b i t o r s : o a r s e n a t e ; A c i t r a t e ; © EDTA. r  ++  -126-  reaction.  Assuming a maximum b i n d i n g o f two M g  m o l e c u l e o f EDTA, i t i s seen t h a t o v e r k%  inhibitory  i o n s by one  of the t o t a l  added was s t i l l p r e s e n t i n t h e form o f f r e e M g EDTA i s p r o b a b l y  + +  + +  ions.  Mg  + +  Thus  i n some manner o t h e r t h a n t h r o u g h  the removal o f M g . ++  P r e v i o u s l y ( 4 6 ) , i t was shown, w i t h t h e i n t e s t i n a l enzyme, t h a t t h e i n h i b i t o r y  effect  o f EDTA was more pronounced  when i t was p r e i n c u b a t e d w i t h DNA, whereas p r e i n c u b a t i o n w i t h c i t r a t e and a r s e n a t e w i t h t h e m e t a l and s u b s t r a t e d i d n o t enhance t h e i r i n h i b i t o r y  power.  with the present f i n d i n g s .  T h i s i s i n complete agreement  I n the l i g h t of k i n e t i c  evidence,  i t i s q u i t e p o s s i b l e t h a t t h e Mg-EDTA complex i n h i b i t s by competing w i t h t h e metal-DNA complex f o r t h e a c t i v e s i t e on the enzyme.  P r e i n c u b a t i o n o f EDTA w i t h M g  + +  presumably i n -  c r e a s e d t h e chance o f f o r m a t i o n o f Mg-EDTA, t h u s i n c r e a s i n g the i n h i b i t o r y  power o f t h e c h e l a t i n g agent.  -127-  SUMMARY 1.  The d e o x y r i b o n u c l e a s e a c t i v i t y o f the i n t e s t i n a l mucosa of the r a t was  2.  Two  studied.  DNases were found t o be p r e s e n t i n an e x t r a c t o f the  mucosal t i s s u e w i t h K r e b s - R i n g e r phosphate  buffer.  These  two enzymes were p h y s i c a l l y s e p a r a b l e on D E A E - c e l l u l o s e columns.  At pH 7 . 8 ,  one DNase was not adsorbed by DEAE-  c e l l u l o s e and c o u l d be r e c o v e r e d by washing w i t h T r i s b u f f e r a f t e r the main peak o f non-adsorbed had passed t h r o u g h .  Another enzyme c o u l d be e l u t e d o f f the 0.15M.  column a t a NaCl c o n c e n t r a t i o n o f about 3.  cationic protein  The f i r s t enzyme was i d e n t i f i e d as DNase I by i t s optimum pH  (6.5-6.8),  i t s r e q u i r e m e n t s f o r b i v a l e n t m e t a l i o n s and  by i t s b e h a v i o u r towards known DNase I i n h i b i t o r s such as EDTA, c i t r a t e and a r s e n a t e .  T h i s enzyme was  capable of  a t t a c k i n g b o t h n a t i v e and h e a t - d e n a t u r e d DNA,  but i t s a c t i -  v i t y towards h e a t e d DNA was o n l y about 20% o f t h a t n a t i v e DNA.  towards  The p r o d u c t s o f the r e a c t i o n v a r i e d from mono-  n u c l e o t i d e s through t o o l i g o n u c l e o t i d e s w i t h a degree p o l y m e r i z a t i o n e q u a l t o o r g r e a t e r t h a n 7. a l l c a r r i e d a m o n o e s t e r i f l e d phosphate 4.  The  of  These p r o d u c t s  group a t the 5 ' - e n d .  second DNase a c t i v i t y was i d e n t i f i e d  t o be o f the DNase  I I type s i n c e i t was most a c t i v e a t pH 3 . 5 not r e q u i r e b i v a l e n t c a t i o n s f o r a c t i v i t y .  "to 4 . 0 and d i d The  products  -128-  were a l s o o f v a r i o u s s i z e s .  The m o n o e s t e r i f i e d  were found a t t h e 3 ' - t e r m i n a l s  o f these  phosphates  products.  5. F o l l o w i n g ion-exchange chromatography, f u r t h e r p u r i f i c a t i o n o f t h e enzymes c o u l d be a c h i e v e d  by p a r t i t i o n on h y d r o x y l -  a.^psatite columns and s t e p w i s e e l u t i o n w i t h phosphate buffers. 6.  The l i n k a g e s p e c i f i c i t y o f t h e two DNases was s t u d i e d by systematic  analyses  of the products o f the r e a c t i o n .  n a t i v e DNA a s s u b s t r a t e and M g  + +  With  a s a c t i v a t o r , DNase I was  shown t o a t t a c k p r e f e r e n t i a l l y t h e l i n k a g e s pApC, pApT and pGpT.  When M n  + +  was u s e d a s a c t i v a t o r , DNase I a t t a c k e d  n a t i v e DNA i n a random f a s h i o n .  The same l a c k o f s p e c i -  f i c i t y was o b s e r v e d when h e a t - d e n a t u r e d DNA was u s e d a s s u b s t r a t e i n t h e presence o f M g . ++  With n a t i v e DNA a s  s u b s t r a t e , t h e i n t e s t i n a l DNase I I showed a p r e f e r e n c e  for  the l i n k a g e s ApCp, GpCp and GpTp. 7. The mechanism o f m e t a l a c t i v a t i o n o f i n t e s t i n a l DNase I was also studied.  The enzyme was found t o be i n h i b i t e d by h i g h  concentrations  o f DNA and m e t a l i o n s .  Optimal a c t i v i t y  appeared t o depend on t h e molar r a t i o o f m e t a l i o n t o DNA phosphorus; and t h i s r a t i o i n t u r n depended on t h e amount o f enzyme p r e s e n t .  These e x p e r i m e n t s suggested t h a t t h e  metal-DNA complex was p r o b a b l y quently, a rate equation  the true substrate.  Conse-  i n terms o f t h e c o n c e n t r a t i o n o f  -129-  the m e t a l l o s u b s t r a t e was  d e r i v e d from a c o n s i d e r a t i o n of  the p e r t i n e n t e q u i l i b r i a : V  K  3E a  s  . q  aKg + K K g Q  A  v = S AS < a + K ) ( aKg + K K )  a K  K  A  ~  K  +  A  0  A S  Data o b t a i n e d from i n i t i a l v e l o c i t y s t u d i e s c a r r i e d out a t v a r y i n g c o n c e n t r a t i o n s o f m e t a l and DNA equation w e l l .  On t h i s b a s i s , i t was  nary complex formed by the c o m b i n a t i o n and DNA 8. The  was  p r o b a b l y an  f i t t e d this rate  suggested t h a t a t e r of enzyme, m e t a l  intermediate.  e f f e c t o f EDTA, c i t r a t e and a r s e n a t e on the  initial  v e l o c i t i e s o f the DNase I r e a c t i o n was a l s o s t u d i e d .  By  r e l a t i n g the e x t e n t of i n h i b i t i o n to the c o n c e n t r a t i o n o f f r e e m e t a l i o n s i n the presence o f v a r y i n g l e v e l s o f c h e l a t i n g agents,  i t was  found t h a t c i t r a t e and  the  arsenate  i n h i b i t e d by s i m p l y removing the m e t a l a c t i v a t o r .  EDTA,  on the o t h e r hand, a p p a r e n t l y i n h i b i t e d the r e a c t i o n i n some o t h e r manner, s i n c e the l e v e l of EDTA c a u s i n g 90$ i n h i b i t i o n was  able to react s t o i c h i o m e t r i c a l l y w i t h only  about 50$ of the added M g . ++  t h a t EDTA p r o b a b l y  A suggested p o s s i b i l i t y i s  i n h i b i t e d t h r o u g h the f o r m a t i o n o f a  metal-EDTA complex which c o u l d compete w i t h metal-DNA f o r the a c t i v e s i t e on the enzyme.  -130-  REFERENCES 1. L a s k o w s k i , li., Adv. Enzymology, 2 9 , 165 ( 1 9 6 ? ) 2 . A r a k i , T., Z. P h y s i o l . Chem., 3 8 , 84 (1903) 3 . K u n i t z , M., J . Gen. P h y s i o l . , 24, 15  (1940)  4 . K u n i t z , M., J . Gen. P h y s i o l . , 3 3 , 3^9 (1950) 5 . S i n s h e i m e r , R.L., and Koerner, J . F . , J . B i o l . Chem., 1 9 8 , 293(1950)  6 . P o t t e r , J . L . , Brown, K.D., and L a s k o w s k i , M., B i o c h i m . B i o p h y s . A c t a , 9 , 150 (1952) 7.  C a t c h e s i d e , D.G., and Holmes, B., Symp. Soc. E x p t l .  I , 225  (1947)  Biol.  8 . Maver, M.E., and Greco, A.E., J . B i o l , . Chem., 181, 8 5 3 , 861  (19^9)  9 . Koerner, J.F., and S i n s h e i m e r , R.L., J . B i o l . Chem., 228, 1039,  1049  (1957)  1 0 . L a u r i l a , U.-R., and L a s k o w s k i , M., J . B i o l . Chem., 228, 49  (1957)  1 1 . Cunningham, L., and L a s k o w s k i , M., B i o c h i m . B i o p h y s . A c t a II,  12. 13.  590  (1953)  Gupta, S., and H e r r i o t t , R.M., A r c h . Biochem. B i o p h y s . , 101,  88 ( 1 9 6 3 )  A l l f r e y , V.G., and M i r s k y , A.E., J . Gen. P h y s i o l . , 3 6 ,  227  (1952)  14.  Shack, J . , J . B i o l . Chem., 2 2 6 , 573 (1957)  15.  H e a l y , J.W., S t o l l a r , D., Simon, M.I., and L e v i n e , L., A r c h . Biochem. B i o p h y s . , 1 0 3 , 4 6 l (1963)  16.  C u r t i s , P . J . , Burden, M.G., and S m e l l i e , R.M.S., Biochem. J . 98,  813  (1966)  1 7 . R i c h a r d s o n , C.G., S c h i l d k r a u t , C.L., and Kornberg, A., C o l d S p r i n g Harbour Symp. Quant. B i o l . , 2 8 , 9 (1963)  -13118. Radding, C M . , Josse, J . , and Kornberg, A., J . B i o l . Chem., 237, 2869 ( 1 9 6 2 ) . 19.  Brody, S., and T h o r e l l , B., Biochim. Biophys. Acta, 2 5 , 579 ( 1 9 5 7 ) .  21.  Shortman, K., and Lehman, I.R., J . B i o l . 2964 ( 1 9 6 4 ) .  Chem., 239,  2 2 . Lehman, I.R., Roussos, G.G., and P r a t t , E.A., J . B i o l . Chem. 237, 819 ( 1 9 6 2 ) . 23.  Dussoix, D., and Arber, W.,  J . Mol. B i o l . ,  5,  37  (1962).  24. Kohn, K.W., S t e i g b i g e l , N.H., and Spears, C.L., £ r p c . N a t l . 4Baa.)Sc|2 ( B J l ^ ) ( 5 2 v 5 U 5 4 ( 1 9 6 5 ) . x  f  25.  Emmerson, P.T., and Howard-Flanders, P., Biochem. Biophys. Res. Commun., 18, 24 (1965),.  26.  Setlow, R.B., and C a r r i e r , W.L., (U.S.), 51, 226 ( 1 9 6 4 ) .  27.  Lynn, S., and Lehman, I.E., J . ' B i o l . Chem., 240, 1287, 1294 ( 1 9 6 5 ) .  Proc. N a t l . Acad.  Sci.  28. Howard-Flanders, P., and Boyce, R.P., G e n e t i c s , 5 ° , 256 (1964). ~ 29.  Meselson, M., J . Mol. B i o l . ,  9, 73^ ( 1 9 6 4 ) .  3 0 . Ochoa, 8., Arch. Biochem. Biophys., 69, 119  (1957).  3 1 . H o l l e y , R.W., Apgar, I . , E v e r e t t , G.A., Madison, J.T., Marquisee,M., M e r r i l l , S.H., Penswick, J.R., and Zamir, A., Science, l4_7, 1462 ( 1 9 6 5 ) . 32.  H a l l , J.B., and Sinsheimer, R.L., J . Mol. B i o l . , (1963) .  6,  115  33.  Yanofsky, C , C a r l t o n , B.C., Guest, J.R., H e l i n s k i , D.R., and Henning, V., Proc. N a t l . Acad. S c i . (U.S.), 51, 266 (1964) .  34.  Sarabhai, A.S., S t r e t t o n , A.O.W., Brenner, S., and B o l l e , A., Nature, 2 0 1 , 13 ( 1 9 6 4 ) .  35.  K u n i t z , M., J . Gen. P h y s i o l . , 3 3 . 363 ( 1 9 5 0 ) .  -13236. L i t t l e , (195D.  J.A., and B u t l e r ,  G.C., J . B i o l .  Chem., 1 8 8 , 6 9 5  37.  S i n s h e i m e r , R.L., a n d K o e r n e r , J.F., S c i e n c e , 114, 42 (1951).  38.  S i n s h e i m e r , R.L., J . B i o l .  3 9 . Overend, W.G.,  Chem., § 0 8 , 445 (1954).  and Webb], .. M., J . Chem. S o c , 2746  (1950).  40. S i n s h e i m e r , R.L., and K o e r n e r , J.F., J . Amer. Chem. S o c , 74, 283 ( 1 9 5 2 ) . 41. S i n s h e i m e r , R.L., J . B i o l .  Chem., 215,-579 (1955).  42. Georgatsos, J.G.,and Antonoglou, 0 . , J . B i o l . Chem., 2 4 l , 2151 ( 1 9 6 6 ) . 43.  Sung, S.C., and Laskowski, M., J . B i o l . Chem., 2 3 7 , 506 (1962).  44.  T a n i u c h i , H., and A n f i n s e n , C.B., J . B i o l . Che., 2 4 l , 4366 ( 1 9 6 6 ) .  45.  D i r k s e n , M.L., and Dekker, C.A., Biochem. Biophys. Res. Commun., 2, 147 ( i 9 6 0 ) .  46.  Lee, C.Y., and Zbarsky, S.H., Can. J . Biochem., 4 5 , 39 (1967). ~~  47.  Lindberg, M.U., Biochim. Biophys. Acta, 82, 237 ( 1 9 6 4 ) .  48.  Lindberg, M.U., J . B i o l . Chem., 241, 1246 ( 1 9 6 6 ) .  49.  McCarty, M., J . Gen. P h y s i o l . , 29, 123 ( 1 9 4 6 ) .  50.  Weissman, N., and F i s h e r , J . , J . B i o l . Chem., 178, 1007 (1949).  51.  Desreaux, V., Hacha, R., and F r e d e r i c q , E., J . Gen. P h y s i o l . 4 J , Suppl. 93 (1962).  52.  Shack, J . , and Bynum, B.S., J . B i o l . Chem., 2^9, 3843  5 3 . Erkama, J . , and Suutarinen, P., A c t a Chem. Scand., 1 3 , 323 ( 1 9 5 9 ) .  (1964)  -133-  54. 55.  Wiberg, J.S., Arch.. Biochem. B i o p h y s . , 7 3 , 337 ( 1 9 5 8 ) . H u r s t , R.O., and B e c k i n g , G.C., Can. J . Biochem. E h y s i p l , ,  41,  469 ( 1 9 6 3 ) .  56.  Bollum, P.J., J . B i o l . Chem., 2 4 0 , 2599  57.  Wiberg, J.S., and Neuman, W.F., A r c h . Biochem. B i o p h y s . , 72,  66  (1965).  (1957).  5 8 . Zubay, G., and Doty, P., B i o c h i m . B i o p h y s . A c t a , 2 9 , 47  59.  (1958).  "Manometric Techniques", (Umbreit, W.W., B u r r i s , R.H., and S t a u f f e r , J.F., e d s . ) , 4 t h ed., p. 1 3 2 , Burgess P u b l . Co.  (1964).  6 0 . P o r a t h , J . , and F l o d i n , P., Nature, I 8 3 , 1657 61.  (1959).  Lowry, O.H., Rosenbrough, N.J., F a r r , A.L., and R a n d a l l , R.J., J . B i o l . Chem., 1 9 3 , 2 6 5 ( 1 9 5 1 ) .  6 2 . K e l l e r , P . J . , Cohen, E., and Neurath, J . B i o l . Chem., 2 3 3 , 344  (1958).  6 3 . L i n d b e r g , U., B i o c h e m i s t r y , 6 , 323 ( 1 9 6 7 ) . 64. B e r g e r , G., and May, P., B i o c h i m . B i o p h y s . A c t a , 1 3 9 , 148  65.  (1967).  K o w l e s s a r , O.D., Altman, K . I . , and Hempelmann, L.H., A r c h . Biochem. B i o p h y s . , 5 2 , 362 ( 1 9 5 * 0 .  6 6 . K o s z a l k a , T.R., S c h r e i e r , K., and Altman, K . I . , B i o c h i m . B i o p h y s . A c t a , lj>, 194 ( 1 9 5 * 0 . 67.  R o b e r t s , M.L., B.Sc. (Honours) T h e s i s , U.B.C, Vancouver (1967).  6 8 . Gavosto, F., B u f f a , F., and M a r i a n i , G., C l i n . 4,  192  (1959).  Chim. A c t a ,  6 9 . Laskowsk^, M., i n "The Enzymes", (Boyer, P.D., L a r d y , H., and Myrback, K., e d s . ) , 2nd ed., V o l . 5 , P. 1 2 3 , Acad. P r e s s , N.Y., ( 1 9 6 l ) . 70.  Felix,  235,  F., P o t t e r , J . L . , and L a s k o w s k i , M., J . B i o l . Chem.,  1150  (I960).  -134-  71.  Tomlinson, R.V., and Tener, G.M., B i o c h e m i s t r y , 2 , 697 (1963).  7 2 . Wyatt, G.R., Biochem. J . , 48, 584  (195D.  7 3 . H o t c h k i s s , R.D., J . B i o l . Chem., 1 7 5 , 315  (1948).  7 4 . P r i v a t de G a r i l h e , M., Cunningham, L., L a u r i l a , U.-R., and L a s k o w s k i , M., J . B i o l . Chem., 2 2 4 , 751 ( 1 9 5 7 ) . 7 5 . L i n , H.J., and C h a r g a f f , E., B i o c h i m . B i o p h y s . A c t a , 1 2 3 , 66  (1966).  7 6 . M a n d e l l , J.D., and Hershey, A.D., A n a l . Biochem., 1, 66 (I960).  77.  Vanecko, S., and L a s k o w s k i , M., 33 B i o l . Chem., 2 3 6 , 3312  (1961).  7 8 . Vanecko, S., and L a s k o w s k i , M., J . B i o l . Chem., 2 3 6 , 1135  (1961).  7 9 . R a l p h , R.K., Smith, R.A., and Khorana, H.G., B i o c h e m i s t r y , 1,  80.  131  (1962).  B e c k i n g , G.C., and H u r s t , R.O., Can. J . Biochem. P h y s i o l . ,  41,  1433 ( 1 9 6 3 ) .  8 1 . J o s s e , J . , K a i s e r , A.D., and Kornberg, A., J . B i o l . Chem., 236,  864 ( 1 9 6 1 ) .  8 2 . Hanson, K.R., B i o c h e m i s t r y , 1,  723  (1962).  8 3 . Thomas, C.A., J r , J . Amer. Chem. S o c , 7 8 , 1861 ( 1 9 5 6 ) . 8 4 . Young, E.T., and S i n s h e i m e r , R.L., J . B i o l . Chem., 2 4 0 , 1274  85.  (1965).  Vanecko, S., and L a s k o w s k i , M., B i o c h i m . B i o p h y s . A c t a , 61,  5^9  (1962).  8 6 . F i s k e , C.H., and SubbaRow, Y., <j. B i o l . Chem., 6 6 , 375 (1925).  8 7 . Shoemaker, D.P., and G a r l a n d , C.W., i n "Experiments i n P h y s i c a l C h e m i s t r y " , p. 1 9 4 , McGraw H i l l ( 1 9 6 2 ) .  -13588. McLean, F.C., and Hastings, A.B., J . B i o l . Chem., 108, 285 (1935). 89. G r e g o i r e , J . , and Gre*goire, J . , B u l l . y±, 291 (1952). 90.  Soc. Chim-. B i o l . ,  G a v a l i e r i , L.F., and Hatch, B., j . Amer. Chem. S o c , 25., 1110 (1953).  91. M i y a j i , T., and G r e e n s t e i n , J.P., Arch. Biochem. Biophy 32, 414 ( 1 9 5 D . 92. Lineweaver, H., and Burk, D., J . Amer. Chem. S o c , 7 6 , 658 (1934). 93. M i c h a e l i s , L., and Menten, M.L., Biochem. Z., 49, 333 (1913). 94. Hanes, C.S., Biochem. J . , 26, 1406 (1932). 95. Shack, J . , J e n k i n s , R.J., and Thomsett, Chem., 2 0 3 , 373 (1952).  J.M., J . B i o l .  96. Dixon, M., and Webb, E.C., i n "Enzymes", p. 440, Acad. Press, N.Y. (1964). 97.  Mildvan, A.S., and Cohn, M., J . B i o l . Chem., 2 4 l , 1178 (1966).  98. C l e l a n d , W.W.,  Ann. Rev. Biochem., 3 6 , 77 (1967).  99. F e i n s t e i n , R.N., and Green, 6 0 , 502 (1956).  F.O., Arch. Biochem. Biophy  100. G i l b e r t , L.M., Overend, W.G., Research, 2, 349 (1951).  and Webb, M., E x p t l .  Cell  101. Hastings, A.B., McLean, F.C., E i c h e l b e r g e r , L., H a l l , J.L., and DaCosta, E., J . B i o l . Chem., 1 0 7 , 3 5 1 (1934).  

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