UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Studies on protamine Ingles, Charles James 1967

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1968_A1 I53.pdf [ 7.38MB ]
Metadata
JSON: 831-1.0093558.json
JSON-LD: 831-1.0093558-ld.json
RDF/XML (Pretty): 831-1.0093558-rdf.xml
RDF/JSON: 831-1.0093558-rdf.json
Turtle: 831-1.0093558-turtle.txt
N-Triples: 831-1.0093558-rdf-ntriples.txt
Original Record: 831-1.0093558-source.json
Full Text
831-1.0093558-fulltext.txt
Citation
831-1.0093558.ris

Full Text

The  U n i v e r s i t y of B r i t i s h  Columbia  FACULTY GF GRADUATE STUDIES PROGRAMME OF THE F I N A L ORAL EXAMINATION FOR THE DEGREE OF DOCTOR OF PHILOSOPHY • of CHARLES JAMES INGLES B.Sc.  (Honours), U n i v e r s i t y  FRIDAY, DECEMBER  o f T o r o n t o , 1964  1 5 , 1967 AT 3:30  P.M.  I N ROOM 2 3 1 , MEDICAL S C I E N C E S BLOCK A  COMMITTEE Chairman: G.H.. D i x o n M. S m i t h W.S. H o a r  I N CHARGE I . McT. Cowan S.H. Z b a r s k y J.F. R i c h a r d s R.A.J. Warren  E x t e r n a l Examiner: W.J. R u t t e r Department of B i o c h e m i s t r y U n i v e r s i t y of Washington S e a t t l e , W a s h i n g t o n , U.S.A.  Research Supervisor:  G.H.  Dixon  STUDIES ON  PROTAMINE  ABSTRACT  S p e r m a t o g e n e s i s i n f i s h i s r a p i d and e x t r e m e form of c e l l u l a r d i f f e r e n t i a t i o n . Protamines, small b a s i c p r o t e i n s a s s o c i a t e d w i t h DNA, a r e p e r h a p s t h e most c h a r a c t e r i s t i c o f t h e s p e r m - s p e c i f i c macromolecules. The i n c o r p o r a t i o n o f a r g i n i n e - H i n t o protamine i n t e s t i s c e l l suspensions from both n a t u r a l l y maturing P a c i f i c salmon (Oncorhynchus spp.) and immature Salmo g a i r d n e r i i , i n w h i c h s p e r m a t o g e n e s i s was i n d u c e d b y hormonal i n j e c t i o n s , p e r m i t t e d a study of the b i o s y n t h e s i s o f p r o t a m i n e . The i n h i b i t o r y a c t i o n o f puromyc i n , c y c l o h e x i m i d e a n d c h l o r a m p h e n i c a l on p r o t a m i n e s y n t h e s i s i n d i c a t e d t h a t p r o t a m i n e was s y n t h e s i z e d b y a r i b o s o m a l system. Methods t o p u r i f y p r o t a m i n e , u t i l i z i n g g e l f i l t r a t i o n , were d e v e l o p e d and p r e l i m i n a r y e x a m i n a t i o n s of the s t r u c t u r e s of protamine from s e v e r a l s p e c i e s of s a l m o n o i d f i s h were begun. P r o t a m i n e was f o u n d t o be p h o s p h o r y l a t e d , a n d the s i t e of t h i s p h o s p h o r y l a t i o n examined. The c h a r a c t e r i z a t i o n o f a number o f t r y p t i c p h o s p h o - p e p t i d e s from protamine i n d i c a t e d that a t l e a s t 4 d i f f e r e n t s e r i n e s i n p r o t a m i n e were p h o s p h o r y l a t e d . T h i s phosp h o r y l a t i o n p r o c e e d e d i n d e p e n d e n t l y o f the amino a c i d i n c o r p o r a t i o n d u r i n g s y n t h e s i s ; however a l l newly i n c o r p o r a t e d s e r y l r e s i d u e s were p h o s p h o r y l a t e d . T h i s p h o s p h o r y l a t i o n may p l a y a r o l e b o t h i n t h e i n t r a c e l l u l a r t r a n s p o r t o f p r o t a m i n e a n d i n t h e c o n t r o l o f gene expression i n these t e s t i s c e l l s .  GRADUATE STUDIES F i e l d of Study:  Biochemistry  B i o c h e m i c a l Methods  Dept. o f B i o c h e m i s t r y  S t r u c t u r e and F u n c t i o n o f P r o t e i n s  G.H. D i x o n W.J. P o l g l a s e  Biochemistry of N u c l e i c A c i d s  G.M. Tener J.F. R i c h a r d s  B i o c h e m i s t r y of Amino A c i d s and P r o t e i n s Biochemistry of Carbohydrates  Biochemistry of L i p i d s  B i o c h e m i s t r y of S t e r o i d s  T h e o r e t i c a l Organic  Chemistry  S.H. Zbarsky  G.I. Drummond J.J.R. Campbell W.J. P o l g l a s e P.D. Bragg C T . Beer J.F. R i c h a r d s P.H. J e l l i n c k G..I. Drummond V . J . O'Donnell P.H. J e l l i n c k A.F. Burton R.E. P i n c o c k  Introductory Genetics  D.T. S u z u k i  Advanced G e n e t i c s  D.T. S u z u k i  PUBLICATIONS  C.J. I n g l e s , J.R. T r e v i t h i c k , M. Smith and G.H.. D i x o n . B i o s y n t h e s i s of Protamine D u r i n g Spermatogenesis i n Salmonoid F i s h , Biochem. B i o p h y s . Res. Commun., 22,627, 1966. J.R. T r e v i t h i c k , C.J. I n g l e s , M. Smith and G.H. D i x o n Protamine B i o s y n t h e s i s D u r i n g Spermatogenesis i n F i s h , P r o c . Fed. Can. B i o l . S o c , 9, 79, 1966. J.R. T r e v i t h i c k , C.J. I n g l e s , and G.H. D i x o n . B i o s y n t h e s i s of Protamine i n T r o u t T e s t i s , . Fed. P r o c , 26, 603, 1967. C.J. I n g l e s , and G.H. D i x o n , P h o s p h o r y l a t i o n of N u c l e a r P r o t e i n s , P r o c . Fed. Can. B i o l . S o c , 10, 117, 1967, C.J. I n g l e s and G.H. D i x o n , P h o s p h o r y l a t i o n of N u c l e a r P r o t e i n s , V I I I n t e r n a t . Cong. Biochem. ( a b s t r a c t ) , Tokyo, 1967. C.J. I n g l e s and G.H. D i x o n , P h o s p h o r y l a t i o n of Protamine D u r i n g Spermatogenesis i n T r o u t T e s t i s , P r o c Nat. Acad. S c i . ( U . S . ) , 58, 1011, 1967.  STUDIES ON PROTAMINE by  B.Sc,  C. JAMES INGLES U n i v e r s i t y o f Toronto, 1964  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 required  to the  standard  THE UNIVERSITY OF BRITISH COLUMBIA December, 1967  In p r e s e n t i n g  for  this  thesis  f u l f i l m e n t of the requirements  an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y  that  the Library  Study.  thesis  shall  I further  make i t f r e e l y  agree that  publication  available  permission  Department  representatives.  of this  w i t h o u t my w r i t t e n  thesis  Columbia,  I agree  f o r r e f e r e n c e and  f o r extensive  copying of this  ^iQC-k^Misfl^  of  jU£&  U  / 0  Columbia  7  It i s understood  for financial  permission.  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, Canada  Date  of British  f o r s c h o l a r l y p u r p o s e s may be g r a n t e d b y t h e Head o f my  D e p a r t m e n t o r b y h.ils  or  in partial  gain  shall  that  copying  n o t be a l l o w e d  STUDIES ON PROTAMINE ABSTRACT Spermatogenesis r a p i d and extreme  i n f i s h i s a remarkable example of a  form of c e l l u l a r d i f f e r e n t i a t i o n .  mines, the s m a l l b a s i c p r o t e i n s a s s o c i a t e d w i t h DNA spermatozoa  Protai n the  of c e r t a i n f i s h , are perhaps the most c h a r a c t e r -  i s t i c of the s p e r m - s p e c i f i c macromolecules. s y n t h e s i s of these unusual p r o t e i n s was  A study of the  i n i t i a t e d u s i n g sus-  pensions of c e l l s from the t e s t e s of both n a t u r a l l y maturing P a c i f i c salmon  (Oncorhynchus  nerii  spermatogenesis had been induced by the i n -  i n which  spp.) and immature Salmo  j e c t i o n of salmon p i t u i t a r y e x t r a c t s .  Protamine,  gaivd-  easily  c h a r a c t e r i z e d by p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s ,  was  only p r e s e n t i n the a c i d e x t r a c t of t e s t i s t i s s u e a t a l a t e stage of spermatogenesis.  The i n c o r p o r a t i o n of a r g i n i n e - H  3  i n t o protamine v/as s t u d i e d i n t h i s t e s t i s c e l l suspension system.  The i n h i b i t i o n of protamine s y n t h e s i s by  puromycin,  c y c l o h e x i m i d e and c h l o r a m p h e n i c o l i n d i c a t e d t h a t i n s p i t e of t h e i r s m a l l s i z e and unusual amino a c i d c o m p o s i t i o n , protamines were s y n t h e s i z e d by a mRNA, tRNA, r i b o s o m a l system Actinomycin D, however, d i d not i n h i b i t t h i s s y n t h e s i s of protamine. Protamines from s e v e r a l d i f f e r e n t s p e c i e s of salmonoid f i s h were p u r i f i e d by a two s t e p chromatography l i z i n g g e l f i l t r a t i o n on Sephadex G-25  procedure u t i  and B i o g e l P-10.  The  amino a c i d compositions of these protamines were compared, the N and C - t e r m i n a l amino a c i d s of the Salmo  gairdnerii  protamine determined, and f r a c t i o n a t i o n of the components of Oncorhynchus  tshawytscha  (chinook salmon) protamine, by  chromatography on both carboxymethyl Sephadex and alumina, attempted. During the late stages of maturation, the t e s t i s c e l l s from S. g a i r d n e r i i ,  which are synthesizing protamine, have  been shown to incorporate phosphate-P and protamine f r a c t i o n s .  32  into both histone  The s i t e of t h i s phosphorylation  of  protamine was i d e n t i f i e d as seryl residues, and a number  of  radioactive phospho-peptides were isolated from the t r y p t i c  digest of protamine-P . 32  The major phospho-peptide was iden-  t i f i e d as val-serP-arg; however phosphorylation of other serine sequences i n protamine was indicated by the presence of  the peptides (ala,serP,arg)arg, (ser,serP)arg and arg-  serP-serP-arg. This phosphorylation of protamine proceeded independently of  the amino acid incorporation during synthesis of protamine;  however, p a r a l l e l incorporation of phosphate-P C* 11  into protamine showed that a l l  32  and serine-  newly incorporated s e r y l  residues were phosphorylated. Since the t o t a l content of ophosphoserine i n protamine f e l l during the l a t e r stages of t e s t i s maturation, a rapid turnover of phosphate i n protamine was indicated.  This view was confirmed by the almost com-  plete absence of phosphate i n protamine prepared from mature spermatozoa of naturally spawning salmonoid f i s h . The phosphorylation of protamine may play a role both i n the in  synthesis and i n t r a c e l l u l a r transport of protamine, and the control of gene expression i n these t e s t i s c e l l s .  iii TABLE OF CONTENTS Page ABSTRACT  i  TABLE OF CONTENTS  i i i  LIST OF TABLES  vi  LIST OF FIGURES  v i i  ACKNOWLEDGEMENT  ix  DEDICATION  X  INTRODUCTION  1  B i o l o g y and Chemistry of Protamine •Meiosis and Spermatogenesis Regulation  o f Gene E x p r e s s i o n  B i o s y n t h e s i s of S p e c i f i c P r o t e i n s PART I  BIOSYNTHESIS AND CHARACTERIZATION OF PROTAMINE  MATERIALS and METHODS  1 7 11 15 21 21  (a) Sources and Husbandry of F i s h  21  (b) C o l l e c t i o n of P i t u i t a r y Glands and P r e p a r a t i o n of P i t u i t a r y E x t r a c t s  22  (c) I n d u c t i o n of Spermatogenesis i n Immature Salmonoid F i s h (d) H i s t o l o g y (e) C h a r a c t e r i z a t i o n of the B a s i c P r o t e i n s by G e l E l e c t r o p h o r e s i s (f) B i o s y n t h e s i s of Protamine i n T e s t i s C e l l Suspensions (i)  I n c u b a t i o n procedures  ( i i ) P u r i f i c a t i o n of r a d i o a c t i v e protamine (g) P u r i f i c a t i o n and C h a r a c t e r i z a t i o n of Protamine  23 24 24 26 26 27 29.  iv (h) F r a c t i o n a t i o n of Protamine (i) Chromatography Sephadex ( i i ) Chromatography  32  on carboxymethyl on alumina  32 32  RESULTS and DISCUSSION  33  (a) I n d u c t i o n of Spermatogenesis i n Salmonoid Fish  33  (b) The B i o s y n t h e s i s of Protamine  43  (c) E f f e c t of I n h i b i t o r s on Protamine Biosynthesis  45  (d) The P u r i f i c a t i o n of Protamine  54  (e) Amino A c i d Composition of P u r i f i e d Protamine  58  (f) Attempted F r a c t i o n a t i o n of Salmonoid Protamines  64  PART I I  PHOSPHORYLATION OF PROTAMINE  73  MATERIALS and METHODS (a) I n c u b a t i o n  74  Procedures  74  (i)  Large s c a l e i n c u b a t i o n s  74  (ii)  I n d i v i d u a l small scale incubations  75  (b) E x t r a c t i o n and P u r i f i c a t i o n of Nuclear Proteins (i)  Large s c a l e p r e p a r a t i o n of phosphoprotamine  ( i i ) P r e p a r a t i o n of phospho-protamine from i n d i v i d u a l i n c u b a t i o n s (c) P u r i f i c a t i o n of Phospho-peptides from Protamine (i)  75 76 77  P u r i f i c a t i o n 'by e l e c t r o p h o r e s i s and chromatography cn paper"  ( i i ) P u r i f i c a t i o n by i o n exchange graphy  75  chromato-  77 79  V  (d) I d e n t i f i c a t i o n and C h a r a c t e r i z a t i o n P u r i f i e d Phospho-peptides (e) Dephosphorylation peptides  of  of Protamine and Phospho-  RESULTS and DISCUSSION  80 80 81  (a) P h o s p h o r y l a t i o n of Histone and Protamine  81  (b) Nature o f the Phospho-protamine  85  Linkage  (c) S i t e of P h o s p h o r y l a t i o n i n Protamine  92  (d) Extent o f P h o s p h o r y l a t i o n of Protamine  98  (e) K i n e t i c s of Protamine P h o s p h o r y l a t i o n and E f f e c t s of I n h i b i t o r s of P r o t e i n S y n t h e s i s POSSIBLE BIOLOGICAL ROLE of PROTAMINE PHOSPHORYLATION BIBLIOGRAPHY  105 112 122  vi LIST OF TABLES Table I  Page Amino a c i d a n a l y s e s of protamine from salmonoid fish  61  II  S u b t r a c t i v e Edman d e t e r m i n a t i o n of the N - t e r m i n a l amino a c i d of 5. g a i v d n e v i i protamine  63  III  Amino a c i d composition of protamine from salmonoid f i s h  71  UV  A n a l y s i s of major t r y p t i c phospho-peptides  97  V  E x t e n t o f p h o s p h o r y l a t i o n of protamine a t d i f f e r e n t stages o f development  99  VI  E f f e c t of c y c l o h e x i m i d e on protamine lation  phosphory-  110  vii LIST OF FIGURES Figure 1.  Page Amino a c i d sequences of protamines from  Clupea  Pallasii  5  2.  T e s t i s weights of 0. k i s u t a h and S. r e c e i v i n g hormonal i n j e c t i o n s  3.  P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s of a c i d e x t r a c t e d p r o t e i n s from S. g a i v d n e v i i t e s t e s a t i n t e r v a l s over p e r i o d of hormonal i n j e c t i o n s  39  Comparisons of a c i d e x t r a c t e d p r o t e i n s from the t e s t e s of hormonally induced and n a t u r a l l y maturing S. g a i v d n e v i i  41  5.  Time course of i n c o r p o r a t i o n of a r g i n i n e - H protamine  46  6.  E f f e c t s of i n h i b i t o r s of p r o t e i n s y n t h e s i s protamine s y n t h e s i s  7.  Gel f i l t r a t i o n p u r i f i c a t i o n of protamine  8.  P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s of samples a t stages i n the g e l f i l t r a t i o n p u r i f i c a t i o n of protamine  59  S e p a r a t i o n of DNS-amino a c i d s by t h i n l a y e r chromatography  65  Carboxypeptidase A and B d i g e s t s of protamine on e l e c t r o p h o r e s i s a t pH 6.5  66  11.  Chromatography Sephadex  of protamine on carboxymethyl  68  12.  Chromatography  of protamine on alumina  13.  G e l f i l t r a t i o n chromatography of P labelled p r o t e i n s from S. g a i v d n e v i i t e s t i s c e l l s  83  14.  Chromatography of p r o t a m i n e - P , protamine and phosphate-P on Dowex 1 x 8 -  86  Chromatography of protamine-P on carboxymethyl Sephadex  87  4.  9. 10.  15. 16.  gaivdnevii  36  into  3  on  . 47 .  56  70  3 2  32  and  phosphate-P  P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s and autoradiography of protamine-P  88  viii 17. 18.  S t a b i l i t y of phospho-protamine and a l k a l i  linkage to acid 90  E l e c t r o p h o r e t o g r a m of p a r t i a l a c i d of p e p t i d e P I  hydrolysate 91  19.  P u r i f i c a t i o n of t r y p t i c phospho-peptides by e l e c t r o p h o r e s i s and chromatography on paper  94  20.  P u r i f i c a t i o n of t r y p t i c phospho-peptides by chromatography on Dowex 50 x 2  95  21.  Gel f i l t r a t i o n chromatography of s e r i n e - C l a b e l l e d p r o t e i n s from S. g a i r d n e r i i t e s t i s c e l l s  101  22.  Autoradiography of e l e c t r o p h o r e t o g r a m of the t r y p t i c d i g e s t of P and s e r i n e - C * l a b e l l e d protamine  103  Time course of i n c o r p o r a t i o n of i n t o protamine  107  1 4  3 2  23. 24.  11  phosphate-P  32  Schematic r e p r e s e n t a t i o n of protamine phosphoryl a t i o n and d e p h o s p h o r y l a t i o n i n t e s t i s c e l l s  119  ix ACKNOWLEDGEMENTS .. I wish t o thank Dr. Gordon H. Dixon f o r h i s c a r e f u l guidance,  encouragement and e n t h u s i a s t i c i n t e r e s t d u r i n g the  course o f t h i s work.  In the i n i t i a l  stages o f these s t u d i e s ,  the generous c o o p e r a t i o n of Dr. M i c h a e l Smith was i n v a l u a b l e , and h i s c o n t i n u i n g i n t e r e s t i n t h i s work has been a p p r e c i a t e d . My a s s o c i a t i o n s w i t h Miss D.L. Kauffman and Drs. J.R. T r e v i t h i c k , D.M. Gibson, J.A. Black, K. Marushige, B.G. J e r g i l , and C.T. Beer, as w e l l as w i t h other members o f the Department of B i o c h e m i s t r y , a t v a r i o u s times i n the course of t h i s work have been most h e l p f u l . I wish t o acknowledge the f i n a n c i a l support o f the N a t i o n a l Research ships.  C o u n c i l of Canada, from whom I r e c e i v e d t h r e e studentThe r e s e a r c h was supported by g r a n t s t o Dr. G.H. Dixon  from both the M e d i c a l Research F i s h e r i e s Research  Board  C o u n c i l of Canada and the  of Canada.  X  DEDICATION to  my wife Helen and my  parents  - 1 INTRODUCTION The years  first  ago  experiments on  " n u c l e i n " , begun n e a r l y  by F r i e d r i c h Miescher have l e d to the modern s t u d i e s  of n u c l e i c a c i d s and  nuclear p r o t e i n s .  i s o l a t e d from the n u c l e i of pus crude p r e p a r a t i o n of DNA. the spermatozoa of Salmo as the work of K o s s e l  Miescher's n u c l e i n ,  c e l l s , can be c o n s i d e r e d  a  His subsequent work (1), u t i l i z i n g salar,  the A t l a n t i c salmon, as w e l l  (2), forms the b a s i s f o r most of  r e c e n t advances i n both the b i o l o g y of and nuclear  100  chemistry  the  of  the  proteins.  The  term " h i s t o n e " i n t r o d u c e d  to the b a s i c p r o t e i n s of the c e l l  i n 1884 nuclei.  by K o s s e l r e f e r s Properly  histones  are b a s i c p r o t e i n s which a t some time are  with DNA  (3).  This d e f i n i t i o n purposely  between h i s t o n e s and  avoids  defined, associated  a distinction  protamine; the l a t t e r are the s m a l l b a s i c  p r o t e i n s , r i c h i n a r g i n i n e , which are found i n the n u c l e i of c e r t a i n spermatozoa, u s u a l l y those of f i s h t i o n between h i s t o n e and molecular  s i z e and  (4).  This  distinc-  protamine i s based on d i f f e r e n c e s i n  a r g i n i n e content,  as w e l l as on the marked  t i s s u e s p e c i f i c i t y of these p r o t e i n s .  Such a d i s t i n c t i o n ,  although a t times r a t h e r a r b i t r a r y , has been observed i n the p a s t , and w i l l s i m i l a r l y be observed i n t h i s t h e s i s . The  B i o l o g y and Chemistry of Protamine The  b i o l o g i c a l and  chemical  aspects  of protamines have  been the s u b j e c t of reviews i n r e c e n t years by F e l i x  (4),  Murray  protamine  (3) and Vendrely and Vendrely  (5).  The  first  - 2 was  isolated  i n 1872  by Miescher  from S. s a l a r  subsequent s t u d i e s , l a r g e l y those o f . K o s s e l  sperm  (1,4)  and  (2), e s t a b l i s h e d  t h a t these protamines occurred  i n the spermatozoa of a number  of d i f f e r e n t s p e c i e s of f i s h .  The  immature t e s t i s c e l l s of  these same s p e c i e s contained only the h i s t o n e type of b a s i c prot e i n s i m i l a r to those p r e s e n t  i n other somatic  though protamines have been found  Clupea  s e v e r a l salmonoid  harengus  Al-  i n the spermatozoa of a  number of f i s h , p r e p a r a t i o n s from both Clupea h e r r i n g ) and  tissues.  pallasii  (Pacific  (European h e r r i n g ) as w e l l as  f i s h , have been the s u b j e c t of the most  i n t e n s i v e examinations (6-8) . The  s m a l l s i z e and unusual  amino a c i d composition  of  the  protamines serve to d i f f e r e n t i a t e them from the other b a s i c proteins present  i n c e l l n u c l e i , e i t h e r h i s t o n e s or b a s i c  p r o t e i n s of ribosomal o r i g i n .  The  remarkably h i g h content  a r g i n i n e , the amino a c i d which c o n s t i t u t e s about two the amino a c i d s p r e s e n t i n protamine, was Kossel  (2).  composition, and  These protamines are very and  leucine  (4,8).  by  i n amino a c i d  a p a r t from a r g i n i n e only c o n t a i n between 5  7 other amino a c i d s ; p r o l i n e or a l a n i n e as  amino a c i d s , and  t h i r d s of  f i r s t described  simple  of  N-terminal  s e r i n e , t h r e o n i n e , v a l i n e , g l y c i n e and In c o n t r a s t the h i s t o n e s found  in  iso-  somatic  t i s s u e s u s u a l l y c o n t a i n most of the 20 n a t u r a l l y o c c u r r i n g amino a c i d s except  tryptophan  Although,  (3,5).  as mentioned, the occurrence  > i n general r e s t r i c t e d  of protamine i s ,  to the spermatozoa of c e r t a i n f i s h , a  s i m i l a r b a s i c p r o t e i n , g a l l i n e , was  obtained  from G a l l u s  gallus  - 3 (rooster) sperm  (9,10).  g a l l i n e , contained  T h i s protamine-type b a s i c p r o t e i n ,  amino a c i d s not p r e s e n t  protamines of h e r r i n g and in  In f a c t , a g r e a t  simple  diversity  spermatozoan n u c l e a r p r o t e i n s e x i s t s among d i f f e r e n t  s p e c i e s and and  salmon.  i n the more  i n many cases  histones  t h i s d i s t i n c t i o n between protamines  is arbitrary.  Not  a l l f i s h c o n t a i n a b a s i c pro-  t e i n i n t h e i r spermatozoa with as simple s i t i o n as the t y p i c a l protamine. carpio  (carp) and  Tinea  b a s i c p r o t e i n , and  Esox  animal  tinea  Among those  (tench)  luoius  an amino a c i d compoexamined  contain a  (pike) a type  Cyprinus  histone-type  intermediate  between h i s t o n e and protamine (5). A b a s i c p r o t e i n from mammalian spermatozoa has e l u s i v e , and sponge-like  i t has  proved  been proposed t h a t these c e l l s c o n t a i n a  k e r a t i n o i d i n t h e i r n u c l e i i n which the DNA  s o l u b l e p r o t e i n s are encased  (11).  Treatment of these  e-mercaptoethanol and deoxyribonuclease  tozoa w i t h  was  and spermaun-  s u c c e s s f u l i n y i e l d i n g an a c i d s o l u b l e b a s i c p r o t e i n (3), and although  a h i g h content  has been noted  of a r g i n i n e i n mammalian spermatozoa  (5), the presence of b a s i c n u c l e a r p r o t e i n s  has  y e t to be demonstrated. That protamine was r a t h e r was Kossel  not a s i n g l e homogeneous p r o t e i n , but  composed of a number of components was  (2).  This heterogeneity  assumed by  became e v i d e n t when the e s t e r  h y d r o c h l o r i d e s were f i r s t prepared  from protamine p i c r a t e (12);  however the s t a r t i n g m a t e r i a l , methods of p r e p a r a t i o n and  of  f r a c t i o n a t i o n a f f e c t e d these e a r l y r e s u l t s c o n s i d e r a b l y .  The  t r u e extent of t h i s h e t e r o g e n e i t y  was  not c l e a r l y  understood  - 4 u n t i l the more r e c e n t work of Ando and h i s a s s o c i a t e s  (6,8).  A number of methods f o r the f r a c t i o n a t i o n of protamine have been used; c o u n t e r - c u r r e n t  (13-15) and paper chromatographic  (4) methods have proved to be only p a r t i a l l y s a t i s f a c t o r y , but the  f r a c t i o n a t i o n of protamine on alumina u s i n g aqueous phos-  phate b u f f e r s f o r e l u t i o n  (14,16) v/as s u c c e s s f u l i n y i e l d i n g  a s i n g l e pure homogeneous s p e c i e s of protamine chromatography  (17).  of e i t h e r f r e e protamine on alumina  By  (6,16) or i t s  t r i n i t r o p h e n y l a t e d d e r i v a t i v e s on carboxymethyl c e l l u l o s e (7, 18),  Ando separated the t h r e e components of protamine from  Clupea of the  pallasii  (Pacific herring).  The amino a c i d  sequences  each of these three components Y l , Y I I , and Z p r e s e n t i n mature t e s t i s  i n the r a t i o 1.0:1.1:0.7, (19) have been  determined r e c e n t l y  (6,7,18) and they are shown i n F i g u r e 1.  A r g i n i n e , the amino a c i d c o m p r i s i n g t w o - t h i r d s of a l l those p r e s e n t , occurs as i s o l a t e d r e s i d u e s and i n " b l o c k s " of up to three and  four.  B l a c k and Dixon  (21) have r e c e n t l y proposed an e v o l u t i o n a r y  pathway t o account f o r both the g r e a t s i m i l a r i t y between the sequences of these t h r e e components of protamine from  Clupea  pallasii  sequence  and the h i g h degree of homology i n amino a c i d  w i t h i n each of the d i f f e r e n t components.  During e v o l u t i o n  these sequences c o u l d have been d e r i v e d from an a r c h e t y p a l pentapeptide a l i n i n e - a r g i n i n e - a r g i n i n e - a r g i n i n e - a r g i n i n e by a s m a l l number of m u t a t i o n a l events i n v o l v i n g repeated p a r t i a l gene d u p l i c a t i o n  (22,23), t o g e t h e r w i t h s i n g l e base  t u t i o n s i n amino a c i d  codons.  substi-  Clupeine Y I  Ala Arg Arg Arg Arg Ser — —  Clupeine Y 11  p  Clupeine  Ala Arg Arg Arg Arg Ser Arg Arg Ala Ser Arg Pro Val Arg Arg Arg Arg Pro Arg Arg —  Z  r0  Arg Arg Arg —  Ser Ser Arg Pro lie Arg Arg Arg Arg Pro Arg Arg Arg Thr Thr Arg Arg Arg Arg Ala Gly Arg Arg Arg Arg  Thr Arg Arg Ala Ser Arg Pro Val Arg Arg Arg Arg Pro Arg Arg —  Val Ser Arg Arg Arg Arg Ala — Arg Arg Arg Arg  Val Ser Arg Arg Arg Arg Ala —  Arg Arg Arg Arg  i  F i g u r e 1 - Comparison of amino a c i d sequences o f protamines from Clupea  pallasii  ( P a c i f i c herring)  (6,7,18).  .The components a r e  a l i g n e d so as t o o b t a i n maximum homology i n amino a c i d sequences between the three components (21).  -  6 -  In the d e t e r m i n a t i o n of the primary mine p r e s e n t i n Clupea  Ando and h i s a s s o c i a t e s  pallasii,  overcame two major problems.  s t r u c t u r e s of the p r o t a -  In order t o proceed w i t h t h e  amino a c i d sequence s t u d i e s on any p r o t e i n , i t i s f i r s t  neces-  sary t o have a pure p r e p a r a t i o n o f a s i n g l e homogeneous p r o t e i n . The  f r a c t i o n a t i o n of these components, which o n l y d i f f e r i n  the case o f YII and Z i n the N-terminal r e g i o n o f 5 o r 6 amino a c i d s , was not p o s s i b l e by methods normally used  with  other p r o t e i n s , f o r both the s m a l l s i z e and predominance of a r g i n i n e bestow on these i n d i v i d u a l components similar properties.  remarkably  However, having s u c c e s s f u l l y  achieved  s e p a r a t i o n o f these components, Ando was then a b l e t o o b t a i n p a r t i a l amino a c i d sequences f o r each a f t e r d i g e s t i o n w i t h t r y p s i n and f r a c t i o n a t i o n of the r e s u l t i n g p e p t i d e s . the high content o f a r g i n i n e i n these protamines a d e t e r m i n a t i o n of primary  structure.  l i m i t e d cleavage of protamine  complicated  However by use o f  by chemical means (6,7) as w e l l  as with the enzyme t h e r m o l y s i n lyticus)  Again  (from Bacillus  thermoproteo-  (18), i t was p o s s i b l e t o o b t a i n the longer o v e r l a p p i n g  sequences necessary f o r the o r d e r i n g of the t r y p t i c p e p t i d e s . That protamines  appear i n t h e t e s t i s c e l l s only  l a t e d u r i n g the course o f spermatogenesis observed by Miescher  (1,4).  appearance o f protamine from Salmo  fontinalis  F e l i x et  al  relatively  i n f i s h had been (24) timed the  more a c c u r a t e l y , o b t a i n i n g  protamine  (brook t r o u t ) only 40 days b e f o r e  spawning but not e a r l i e r , and s i m i l a r s t u d i e s of Ando and Hashimoto  (25) w i t h Salmo  gaivdnevii  (rainbow  t r o u t ) confirmed  -  7 -  t h i s r e s u l t , as d i d the h i s t o c h e m i c a l s t u d i e s of A l f e r t (26) on Onoorhynchus  tshawytscha  (chinook salmon)'.  These  s t u d i e s demonstrated t h a t the n u c l e a r p r o t e i n was transformed  a t a l a t e stage of spermatogenesis  type t o the protamine (27) suggested  type.  latter  suddenly  from the h i s t o n e  The c y t o c h e m i c a l r e s u l t s of Block  t h a t i n L o l i g o opalescens  (squid) the h i s t o n e s ,  which o c c u r r e d i n somatic c e l l s and e a r l y spermatogenic  cells,  were r e p l a c e d s u c c e s s i v e l y by d i f f e r e n t b a s i c p r o t e i n s , f i r s t an a r g i n i n e - r i c h h i s t o n e , and f i n a l l y  two protamines.  A  s i m i l a r sequence may take p l a c e i n some s p e c i e s , may stop midway i n o t h e r s , and may not occur a t a l l i n a t h i r d group ( 5 ) . In  any event, t h i s change, i f i t does occur, takes p l a c e l a t e  in  spermatogenesis  w e l l a f t e r completion  c e l l s a r e a t the spermatid  of m e i o s i s w h i l e  stage.  M e i o s i s and Spermatogenesis The process of m e i o s i s and the subsequent are l i t t l e understood.  spermiogenesis  They r e p r e s e n t an extreme example of  c e l l d i f f e r e n t i a t i o n , a p r o c e s s , the b i o c h e m i c a l a s p e c t s of which a r e o n l y now being s u b j e c t e d t o i n t e n s i v e The  examination.  extent of t h i s d i f f e r e n t i a t i o n which r e s u l t s i n a c e l l so  u n i q u e l y s p e c i a l i z e d f o r the packaging  and d e l i v e r y of the  g e n e t i c i n f o r m a t i o n t o the egg i n f e r t i l i z a t i o n was of course f i r s t realized  i n the s t u d i e s of l i g h t m i c r o s c o p i s t s (28)..  Although g r e a t d i f f e r e n c e s i n s i z e , s t r u c t u r e and d e t a i l s of development do e x i s t among spermatozoa of v a r i o u s s p e c i e s (29) a g e n e r a l i z e d d i s c u s s i o n of the o v e r a l l process i s of v a l u e a t  - 8 this point.  The v a s t numbers of spermatozoa  produced by an  organism come from p r e c u r s o r c e l l s known as spermatogonia. These c e l l s d i v i d e r e p e a t e d l y by m i t o s i s , and a number  undergo  m e i o t i c or r e d u c t i o n d i v i s i o n s t o form, from the s i n g l e diploid cell,  four haploid c e l l s .  The d i p l o i d c e l l  original  entering  the m e i o t i c phase i s c a l l e d a primary spermatocyte, and i t s two daughter c e l l s  secondary spermatocytes.  The f o u r  final  products of m e i o s i s , now h a p l o i d , a r e c a l l e d spermatids. spermatids undergo  a number o f complex morphogenetic  t h a t produce the mature spermatozoa.  These changes  These  changes include  an i n t r a c e l l u l a r m i g r a t i o n of m i t o c h o n d r i a t o a p o s i t i o n behind the n u c l e u s .  During t h i s p e r i o d the spermatid c e l l  elon-  g a t e s , r e d u c i n g i t s cytoplasm and producing one or more f l a g e l l a . W i t h i n the n u c l e u s , the chromatin becomes more compact so t h a t the mature spermatozoan  n u c l e i a r e o f t e n only a s m a l l  fraction  of the s i z e of the parent spermatogonial n u c l e i . There are a number of d e t a i l e d accounts of the h i s t o l o g y of the t e s t e s of salmonoid f i s h a t v a r i o u s phases sexual m a t u r a t i o n (26,30,31).  i n this  The completely immature or i n -  f a n t i l e t e s t i s comprising about 0.1 p e r c e n t of the body weight (30) c o n s i s t s of s m a l l c l o s e l y - p a c k e d c y s t s or nests of the p r i m i t i v e germ c e l l s ,  spermatogonia.-  These c y s t s i n groups of  2 t o 6 or more are separated i n t o l o b u l e s , the w a l l s of which although o f t e n i l l - d e f i n e d , are made up of c o n n e c t i v e t i s s u e and blood v e s s e l s .  An i n c r e a s e i n the number of c e l l s i n the  c y s t s , due t o m i t o t i c d i v i s i o n s of the spermatogonia, i s the first  evidence of spermatogenesis.  With i n c r e a s i n g numbers  - 9 these c e l l s become s m a l l e r .  These c e l l s ,  now  designated p r i -  mary spermatocytes, undergo the two m e i o t i c d i v i s i o n s  forming  i n t u r n secondary  cells,  spermatocytes,  then spermatids, the  which w i t h no f u r t h e r d i v i s i o n s , are transformed i n t o spermatozoa. Although the m i c r o s c o p i c d e t a i l s of spermatogenesis  and  i t s c y c l e are q u i t e w e l l known, the u n d e r l y i n g m o l e c u l a r mechanisms are not understood a t a l l . t h i s area may  be i n p a r t due  The  l a g of p r o g r e s s i n  to the f a c t t h a t these complex  processes of d i f f e r e n t i a t i o n are extremely d i f f i c u l t t o s u b j e c t to e x p e r i m e n t a l a n a l y s i s . in vitro available  Only r e c e n t l y have methods f o r the  c u l t i v a t i o n of c e r t a i n mammalian germ c e l l s been (32), and these w i l l ho doubt f a c i l i t a t e  further  s t u d i e s of the s u b c e l l u l a r events i n v o l v e d i n spermatogenesis. The development of the germ c e l l s ,  i n mammalian s p e c i e s  at any r a t e , i s under the c o n t r o l of the a n t e r i o r p a r t of the p i t u i t a r y gland, the adenohypophysis, t r o p h s hormones, f o l l i c l e i z i n g hormone (LH)  (33).  which produces  two  gonado-  s t i m u l a t i n g hormone (FSH) and  lutein-  The a c t i v i t y of t h i s g l a n d i n the  case of salmonoid s p e c i e s i s i n f l u e n c e d by p h o t o p e r i o d (34). Although both f o l l i c l e s t i m u l a t i n g and l u t e i n i z i n g  activities  have been w e l l c h a r a c t e r i z e d i n the h i g h e r v e r t e b r a t e s (35,36), the presence and r o l e of both of these i n t e l e o s t f i s h i s a t present u n c e r t a i n (37).  Both f o l l i c l e  i z i n g a c t i v i t i e s can be demonstrated  s t i m u l a t i n g and  lutein-  i n p i t u i t a r y glands of  t e l e o s t f i s h u s i n g the mammalian assays  (38-40); however the  p h y s i o l o g i c a l r o l e of both of these i n f i s h has y e t to be  - 10 c l e a r l y shown.  In a number of s t u d i e s , mammalian LH, but not  FSH, gave r i s e t o the p h y s i o l o g i c a l response i n f i s h normally a s s o c i a t e d w i t h the two separate gonadotrophins  (41-43).  It is  g e n e r a l l y accepted t h a t the p i t u i t a r y gonadotrophins a c t on the S e r t o l i and L e y d i g c e l l s of the t e s t i s ;  the r e s u l t i n g  g e n e s i s , perhaps mediated by the n u c l e o t i d e ,  steroido-  adenosine-3',5'-  c y c l i c phosphate  (44), b e i n g r e s p o n s i b l e f o r the subsequent  spermatogenesis.  T e s t o s t e r o n e i n l a r g e amounts can i n f a c t  m a i n t a i n spermatogenesis trophins  i n the absence of p i t u i t a r y gonado-  (45). These s t e r o i d o g e n i c c e l l s p r e s e n t i n the t e s t e s  of mammalian s p e c i e s appear t o have t h e i r c o u n t e r p a r t i n the testes of t e l e o s t f i s h .  The endocrine t i s s u e s of these t e s t e s  are e i t h e r arranged as i n t e r s t i t i a l aggregates or d i s p o s e d as l o b u l e boundary  cells  (37). The presence of a number of andro-  gens i n the t e s t e s and blood of the P a c i f i c noted  (46,47).  salmon has been  In p a r t i c u l a r t h e r e i s a h i g h c o n c e n t r a t i o n of  1 1 - k e t o t e s t o s t e r o n e i n the blood of these salmon, as an androgen  has been suggested  and i t s r o l e  (48,49).  From the f o r e g o i n g c o n s i d e r a t i o n s i t w i l l be apparent t h a t spermatogenesis  i n f i s h i s an example of a hormonally  trolled cellular differentiation.  This d i f f e r e n t i a t i o n ,  con-  now  w e l l c h a r a c t e r i z e d m o r p h o l o g i c a l l y , has o n l y i n a few cases been s u b j e c t f o r b i o c h e m i c a l i n v e s t i g a t i o n s .  Of the many  complex changes o c c u r r i n g d u r i n g spermatogenesis  i n f i s h , the  replacement of h i s t o n e s by protamine was perhaps most e a s i l y recognized  (1,2) and t h e r e f o r e i t has been s u b j e c t t o a more  intensive investigation.  Although the t e s t e s of salmonoid  fish  have long been a source f o r the p r e p a r a t i o n enzymatic processes i n v o l v e d  of DNA  (50,51),  i n the s y n t h e s i s of DNA  t i s s u e s have only been s t u d i e d to a l i m i t e d extent. pyrimidine  bases are i n c o r p o r a t e d  mature t e s t e s side-5  1  (52)  and  phosphates and  (53-56).  Studies  polymerase and  the s y n t h e s i s  RNA  being  of  i n these Purine  and  nucleo-  a l s o been examined .  initiated  i n an  effort  enzymes of n u c l e i c a c i d s y n t h e s i s ,  polymerase, p r e s e n t  and  partially  of n u c l e o s i d e s  polyphosphates has  are only now  to c h a r a c t e r i z e the two  i n t o the DNA  the  i n these t i s s u e s  DNA  (57).  A number of enzymes which degrade n u c l e i c a c i d s have a l s o been demonstrated. (58), and and  Salmon t e s t e s are extremely r i c h i n DNase I I  the presence of t h i s enzyme together  phosphatases  i s the s y n t h e s i s preclude  with  diesterases  (58,59) i n a t i s s u e , the major r o l e of which of DNA,  a degradative  i s somewhat unexpected, and  hence  r o l e f o r c e r t a i n of these enzymes  A f u r t h e r c h a r a c t e r i z a t i o n of both RNA  and  protein  may  (60).  species  present  i n the t e s t i s c e l l s at d i f f e r e n t stages of spermato-  genesis  w i l l a i d i n an understanding of t h i s example of  cellular  differentiation. Regulation The and  of Gene  important g e n e r a l i z a t i o n s of chromosome constancy  l a t e r of DNA  e n t i a t i o n was and  Expression  constancy  (62)  (61)  l e d to the view t h a t c y t o d i f f e r -  the r e s u l t of a v a r i a b l e gene e x p r e s s i o n .  Stedman  Stedman having observed t h a t the b a s i c p r o t e i n s of salmon  sperm and  fowl e r t h r o c y t e  and  l i v e r were d i f f e r e n t , proposed  t h a t these b a s i c p r o t e i n s a s s o c i a t e d w i t h DNA  could e x h i b i t a  - 12 cell  s p e c i f i c i t y and might thereby p l a y a r o l e i n the r e g u l a -  t i o n of gene e x p r e s s i o n (63,64).  The experiments of Huang and  Bonner (65) p r o v i d e d some i n d i r e c t support f o r t h i s h y p o t h e s i s , for  h i s t o n e s were found t o i n h i b i t the a c t i o n of DNA-dependent  RNA  polymerase  of  DNA,  in vitro.  Chromatin,  now  shown t o be composed  h i s t o n e s , non-histone p r o t e i n and RNA  (66), when i s o -  l a t e d i n the n a t i v e s t a t e , supports the s y n t h e s i s of RNA presence of an exogenous RNA  polymerase  (67).  i n the  By c o u p l i n g  system t o a r e a c t i o n mixture f o r p r o t e i n s y n t h e s i s , and  this  by  u s i n g an immunological assay f o r examination of the p r o t e i n p r o d u c t s , Bonner et al the  in vitro  (67) have shown t h a t chromatin d i r e c t s  s y n t h e s i s of mRNA and hence p r o t e i n s c h a r a c t e r -  i s t i c of the t i s s u e i n v i v o . r e s t r i c t e d range of DNA  I t was  suggested t h a t o n l y a  sequences were a v a i l a b l e f o r t r a n s c r i p -  t i o n i n chromatin, the m a j o r i t y being "masked" by b a s i c Chromatin and s y n t h e t i c mixtures of h i s t o n e and DNA insoluble i n dilute salt r e s t r i c t i o n of the DNA  are r a t h e r  (68,69) and the p o s s i b l i t y t h a t the  template observed i n such chromatin or  n u c l e o h i s t o n e p r e p a r a t i o n s was  due i n f a c t o n l y t o the p r e c i p i -  t a t i o n and i n a c c e s s i b l i t y of such s u b s t r a t e chromatin t o polymerase the  RNA  proteins.  has been r a i s e d  synthesized i n v i t r o  (68).  RNA  Hybridization studies with  from chromatin p r e p a r a t i o n s i n -  d i c a t e d , however, t h a t only a r e s t r i c t e d number of the chromosomal DNA  sequences was  t r a n s c r i b e d by RNA  and t h i s r e s t r i c t i o n of DNA  (70),  template a c t i v i t y i n chromatin  subsequently shown to be o r g a n - s p e c i f i c of  polymerase  (71).  was  Thus the nature  the "masking" m a t e r i a l i s such t h a t chromatin can be  readily  .- 13 i s o l a t e d w i t h the s p e c i f i c p a t t e r n of "masking" The hypothesis  undisturbed.  of Stedman and Stedman envisaged  that  d i f f e r e n t h i s t o n e s p e c i e s would i n t e r a c t with d i f f e r e n t gene loci,  and hence o f f e r a means f o r the s e l e c t i v e c o n t r o l of  e x p r e s s i o n of these gene l o c i .  However there are many d i f -  f e r e n t genes to be r e p r e s s e d and s p e c i e s of h i s t o n e s cificity  (66).  the  only a s m a l l e r number of  Furthermore the marked t i s s u e spe-  of these b a s i c p r o t e i n s i s not a u n i v e r s a l f e a t u r e ,  for  i n g e n e r a l , c e l l s from widely d i f f e r e n t t i s s u e s w i t h i n  one  s p e c i e s and among d i f f e r e n t s p e c i e s e x h i b i t the same com-  plement of h i s t o n e molecules  (72,73).  A s e r i e s of r e a c t i o n s has been observed are a c e t y l a t e d (74), methylated 77).  i n which h i s t o n e s  (74) and phosphorylated  (75-  These more s u b t l e m o d i f i c a t i o n s of h i s t o n e s t r u c t u r e  o f f e r a means whereby the i n t e r a c t i o n of the b a s i c p r o t e i n s w i t h DNA  may  be s e l e c t i v e l y a l t e r e d to p r o v i d e a b a s i s f o r  v a r i a b l e gene e x p r e s s i o n . (78), t h a t i n pea  Huang and Bonner have r e p o r t e d  s e e d l i n g s , a unique s p e c i e s of RNA  high content of d i h y d r o u r i d y l i c a c i d i s found histones.  with a  complexed with  Such a histone-RNA complex c o u l d p r o v i d e  another  means f o r c o n t r o l of gene e x p r e s s i o n , f o r ' a l t h o u g h the number of  d i f f e r e n t s p e c i e s of h i s t o n e s i s not s u f f i c i e n t to  o f f e r the r e q u i r e d c o n t r o l , a complexing of these molecules  alone  histone  with s p e c i f i c RNAs which i n t u r n b a s e - p a i r with  and  hence r e c o g n i z e d i f f e r e n t r e g i o n s of the genome i s e n t i r e l y possible  (78-80).  In yet another  model f o r the c o n t r o l of  h i s t o n e s as gene r e p r e s s o r s , i t was proteins  suggested  (81) or other n u c l e a r p o l y a n i o n s  t h a t phospho-  (82) c o u l d complex  - 14 with h i s t o n e s and  thus weaken t h e i r b i n d i n g to DNA,  s e l e c t i v e t r a n s c r i p t i o n of the DNA A v a r i e t y of m a n i f e s t a t i o n s r e g u l a t i o n of RNA  enabling  template.  of the d i f f e r e n t i a l  genetic  s y n t h e s i s has been found i n the c e l l s  h i g h e r organisms as w e l l as i n b a c t e r i a .  of  Regulator-operator  c o n t r o l systems which have been d e s c r i b e d i n b a c t e r i a i n v o l v e the modulation of RNA  s y n t h e s i s a t s t r u c t u r a l gene l o c i  r e p r e s s o r substances encoded at other gene l o c i  by  (83).  In the  p o l y t e n e chromosomes of d i p t e r a n l a r v a e , c y t o c h e m i c a l  studies  revealed that c e r t a i n regions c e n t r e s of h i g h l y a c t i v e RNA s e v e r a l s p e c i e s one  RNA  synthesis  (84).  In females of  of the sex chromosomes i n each c e l l r e -  mains h e t e r o p y c n o t i c l a c k of any  (puffs) i n the chromosomes are  and  i n a c t i v e during interphase  (85).  s y n t h e s i s i n both mature spermatozoa  the n u c l e a t e d a v i a n e r y t h r o c y t e  (87) has  The  (86)  and  a l s o been noted.  It  would appear then t h a t a h i e r a r c h y of g e n e t i c r e g u l a t o r y mechanisms i s l i k e l y t o e x i s t i n higher forms. c o n t r o l systems, each w i t h i t s own substances, served  may  A number of  appropriate  type of  different repressor  be r e q u i r e d to o f f e r the wide v a r i e t y i n ob-  l e v e l s of r e g u l a t i o n .  There are at l e a s t two p r o t e i n , protamine, p r e s e n t Although these  two  probable  f u n c t i o n s f o r the b a s i c  i n the mature spermatozoa of  f u n c t i o n s may  not be at a l l s e p a r a b l e ,  fish. the  protamines are l i k e l y to be both i n v o l v e d i n the packaging of the DNA  molecules i n the sperm head and  r e s p o n s i b l e i n some  p a r t f o r the t o t a l r e p r e s s i o n of gene e x p r e s s i o n mature spermatozoa.  i n these  Both the c y t o l o g i c a l s t u d i e s which d e t a i l  the p r o g r e s s i v e condensation of the chromatin d u r i n g maturation  sperm  (26,30,31), and X-ray d i f f r a c t i o n s t u d i e s on n a t i v e  (20,88) and r e c o n s t i t u t e d i n t a c t sperm heads  (89) nucleoprotamine, as w e l l as on  (20,88,90) i n d i c a t e t h a t i n the n u c l e i of  mature spermatozoa the nucleoprotamines are compact, line structures.  crystal-  I t has been suggested (20,88) t h a t the  protamine moiety runs h e l i c a l l y i n the minor groove of the double h e l i x .  The r a t i o of DNA  i n spermatozoa  i s close to unity  phosphate r e s i d u e s of DNA dues of protamine.  DNA  phosphate to protamine a r g i n i n e (4,25), i n d i c a t i n g t h a t the  are n e u t r a l i z e d by the a r g i n y l  By r e d u c i n g DNA-DNA e l e c t r o s t a t i c  resi-  repul-  s i o n s , protamine presumably would f a c i l i t a t e the f o l d i n g of the DNA  i n t o the compact s t r u c t u r e of the spermatozoan n u c l e u s .  Both the s y n t h e t i c homopolymer, p o l y l y s i n e , and protamine, are e f f e c t i v e i n h i b i t o r s of DNA-dependent RNA T h i s evidence, o b t a i n e d i n v i t r o , RNA  as w e l l as the l a c k of any  s y n t h e s i s i n mature spermatozoa  complexing of DNA  s y n t h e s i s (91).  (86) suggests t h a t the  w i t h protamine which occurs a t a l a t e stage  i n spermatogenesis s e r v e s s i m u l t a n e o u s l y t o package  the  DNA  i n the sperm head and to assure' that i t s genes are c o m p l e t e l y "shut o f f " . fertilization  The subsequent disappearance of protamine  after  (92) corresponds w i t h the r e s t o r a t i o n of g e n e t i c  i n f o r m a t i o n t o an a c t i v e s t a t e d u r i n g e a r l y embryonic development. B i o s y n t h e s i s of S p e c i f i c  Proteins  I n t e r e s t i n the s y n t h e s i s of protamine d u r i n g  spermato-  - 16 genesis The  i n the t e s t i s c e l l s of f i s h stems from s e v e r a l f e a t u r e s .  s y n t h e s i s of protamine a t a d e f i n i t e stage l a t e i n spermato-  genesis  represents  one  biochemical  manifestation  f e r e n t i a t i o n which r e s u l t s i n the formation tozoa.  Furthermore, protamine and  of the  dif-  of a mature sperma-  the other b a s i c p r o t e i n s i n  c e l l n u c l e i have themselves been i m p l i c a t e d as having r o l e i n c o n t r o l of these same processes  of  an a c t i v e  differentiation.  F i n a l l y , the s y n t h e s i s of s p e c i f i c p r o t e i n s , such as protamine, i n c e l l - f r e e systems o f f e r s a u s e f u l experimental  t o o l f o r the  d e l i n e a t i o n of c e r t a i n f e a t u r e s of the mechanism of p r o t e i n synthesis. The  dramatic progress  i n r e c e n t years  has  of the f i e l d  of molecular  l e d to an understanding of the  mechanism of p r o t e i n s y n t h e s i s  (93).  a c t i v a t i o n of i n d i v i d u a l amino a c i d s  T h i s process (94)  and  to amino a c i d s p e c i f i c t r a n s f e r RNAs (tRNAs). fulfill  general involves  their transfer These tRNAs  an adaptor r o l e b r i n g i n g the amino a c i d s to the r i b o -  somes (95), where the sequence of the n u c l e o t i d e s RNA  biology  i n a messenger  (mRNA) determines the sequence of amino a c i d s i n the nascent  polypeptide  chain  (96).  The  a c t u a l p e p t i d e bond  formation  takes p l a c e on t h i s ribosome-mRNA-aminoacyl tRNA complex, r o l e of the ribosomes probably separate m o i e t i e s These g e n e r a l  being  to g i v e each of these  the c o r r e c t alignment f o r r e a c t i o n  (97).  f e a t u r e s of p r o t e i n s y n t h e s i s have f o r the most  p a r t been d e l i n e a t e d i n experiments u t i l i z i n g c e l l - f r e e The  a v a i l a b i l i t y of a v a r i e t y of w e l l d e f i n e d  nucleotides  the  systems.  synthetic poly-  (9 6) which i n t u r n d i r e c t the s y n t h e s i s of  similarly  - 17 w e l l d e f i n e d amino a c i d homo- o r heteropolymers  has been i n -  strumental i n g a i n i n g an understanding of the r e l a t i o n s h i p between the n u c l e o t i d e codons and s p e c i f i c amino a c i d s . other hand the attempts proteins i n v i t r o  On the  to synthesize n a t u r a l l y occurring  u t i l i z i n g n a t u r a l l y o c c u r r i n g mRNA templates  have been beset with many problems, have l e d t o the r e f l e c t i o n  and these  (98) t h a t such  difficulties  mRNAs appear t o be  as e l u s i v e as the renowned " S c a r l e t Pimpernel" (99). Of the p r o t e i n s p r e s e n t i n the c e l l n u c l e i of mature spermatozoa of f i s h , protamine Stedman and Stedman proposed components, protamine,  i s c e r t a i n l y the major s p e c i e s .  t h a t sperm n u c l e i c o n s i s t of three  n u c l e i c a c i d and chromosomin  (100).  T h i s l a t t e r component was c o n s i d e r e d t o be p r o t e i n i n nature but c o u l d amount t o o n l y a s m a l l p r o p o r t i o n of the t o t a l n u c l e a r m a t e r i a l f o r P o l l i s t e r and M i r s k y r e p o r t e d (101) t h a t nucleoprotamine  (DNA p l u s protamine)  mass of t r o u t sperm n u c l e i .  accounted  f o r 91 p e r c e n t of the  C y t o l o g i c a l s t u d i e s (26,30,31) have  indicated that there i s l i t t l e  cytoplasm i n the mature sperma-  tozoa, or i n c e l l s a t the e a r l i e r spermatid stage when is  synthesized.  protamine  I t i s probable t h e r e f o r e t h a t t h e major pro-  p o r t i o n of the p r o t e i n s y n t h e t i c  a c t i v i t y of t e s t i s c e l l s a t  the spermatid stage of m a t u r a t i o n would be d i r e c t e d the s y n t h e s i s of t h i s s i n g l e type of p r o t e i n .  towards  A study of pro-  t e i n s y n t h e s i s i n these c e l l s o r i n c e l l - f r e e systems d e r i v e d from them might t h e r e f o r e permit an examination sis  of one s p e c i f i c type of p r o t e i n , protamine.  of the syntheCell-free  systems a c t i v e i n the s y n t h e s i s of other s p e c i f i c p r o t e i n s  - 18 have been s t u d i e d , f o r example a c e l l - f r e e e x t r a c t d e r i v e d from r e t i c u l o c y t e s has been u t i l i z e d synthesis and  (102,103), and  systems d e r i v e d from p a n c r e a t i c  p i t u i t a r y t i s s u e (105)  s u l i n and  i n a study of hemoglobin  are a c t i v e i n the s y n t h e s i s of i n -  a d r e n o c o r t i c o t r o p h i c hormone (ACTH) r e s p e c t i v e l y .  However, the s y n t h e s i s of protamine i n the t e s t i s c e l l s f i s h o f f e r s c e r t a i n advantages. 30 or 35 amino a c i d r e s i d u e s RNA  (104)  The  s m a l l s i z e of protamine,  (4), suggests t h a t a messenger  f o r such a p r o t e i n , i f m o n o c i s t r o n i c ,  only 90 to 100  nucleotides.  protamine where i t occurs  of  The  might c o n s i s t of  high content  of a r g i n i n e i n  i n " b l o c k s " of up t o f o u r  residues  (see F i g u r e 1) suggests t h a t such a mRNA should possess r e peating for  s e c t i o n s of n u c l e o t i d e sequence c o n t a i n i n g the codon(s)  arginine.  An  v a t i n g enzymes and  increased concentration of t r a n s f e r RNA  of amino a c i d a c t i -  s p e c i f i c f o r a r g i n i n e might  a l s o be found i n t e s t i s c e l l s s y n t h e s i z i n g protamine. The  in vitro  experiments w i t h  synthetic  polynucleotides  which d i r e c t the s y n t h e s i s of s p e c i f i c amino a c i d polymers (96, 106)  and  the use  of t r i n u c l e o t i d e templates of known se-  quence v/hich s t i m u l a t e the b i n d i n g of aminoacyl tRNA to r i b o somes (107)  have made p o s s i b l e the i d e n t i f i c a t i o n of the  codons f o r a l l 20 n a t u r a l l y o c c u r r i n g amino a c i d s .  RNA  In many  cases the degeneracy of t h i s t r i p l e t code i s apparent, f o r example 6 d i f f e r e n t codons have been assigned arginine.  These codons f o r a r g i n i n e f a l l  (a) CpGpU, CpGpC, CpGpA and 109).  CpGpG;  t o the amino a c i d  i n t o tv/o main groups:  (b) ApGpA and ApGpG  (108,  There i s a s i m i l a r m u l t i p l i c i t y of tRNAs f o r many of  - 19 the d i f f e r e n t amino a c i d s globin synthesis  (96, 110). S t u d i e s on r a b b i t hemo-  have i n d i c a t e d t h a t two s e p a r a b l e types o f  tRNA, s p e c i f i c f o r l e u c i n e and which correspond t o d i f f e r e n t codon groups, t r a n s f e r l e u c i n e protein  (111) .  positions  into different positions  A s i m i l a r t r a n s f e r of a r g i n i n e  i n this  into different  i n r a b b i t hemoglobin by d i f f e r e n t arginyl-tRNAs has  a l s o been r e p o r t e d  (112) .  Although the g e n e t i c  code i s  e s s e n t i a l l y u n i v e r s a l , t r a n s f e r RNA from one organism  differs  from t h a t of others i n the r e l a t i v e response t o c e r t a i n codons (113). Apart from the s t u d i e s  on hemoglobin  (111, 112) and i n -  d i r e c t evidence from a study of amino a c i d replacements i n several proteins  (23, 114), l i t t l e  z a t i o n of the v a r i o u s acids  i n the s y n t h e s i s  multiple  i s known about the u t i l i -  codons f o r many of the amino  of n a t u r a l p r o t e i n s .  Recent  investi-  g a t i o n s w i t h s a t e l l i t e tobacco n e c r o s i s v i r u s i n d i c a t e t h a t the RNA of t h i s v i r u s c o n t a i n s j u s t s u f f i c i e n t i n f o r m a t i o n for  t o code  i t s coat p r o t e i n which c o n t a i n s approximately 400 amino  acid residues  (115, 116). The a c c u r a t e i d e n t i f i c a t i o n of RNA  codons and o f i n i t i a t i n g and t e r m i n a t i n g  sequences i n mRNA  molecules may be p o s s i b l e w i t h such a system. A c h a r a c t e r i z a t i o n of the protamine s y n t h e s i s  system i n  t e s t i s t i s s u e of f i s h was i n i t i a t e d f o r i t might permit an e l u c i d a t i o n of v a r i o u s thesis.  d e t a i l s i n the mechanism of p r o t e i n  syn-  In the course of these i n v e s t i g a t i o n s c e r t a i n an-  c i l l a r y problems developed.  I t was apparent t h a t  spermato-  genesis i n f i s h was a marked example of c e l l u l a r d i f f e r e n t i a t i o n ,  -  and  the  synthesis  of  specific  a definite  stage i n t h i s  interest.  Furthermore, the  reaction  demonstrated  for  i t could  and  intracellular  control  of  play  this  an  20  -  proteins  s u c h as  differentiation  was  phosphorylation  protamine,  therefore  at  of  of protamine,  a  i n these s t u d i e s , a t t r a c t e d a t t e n t i o n , important  role  not  only  t r a n s p o r t of protamine, but  process  of  differentiation  i n the also  synthesis in  in testis  the tissue.  - 21 PART I BIOSYNTHESIS AND CHARACTERIZATION OF PROTAMINE MATERIALS and METHODS (a)  Sources and Husbandry  of F i s h  Over the p e r i o d of these i n v e s t i g a t i o n s from 1964 t o 1967 s e v e r a l d i f f e r e n t s p e c i e s o f salmonoid f i s h have been used.  In  p r e l i m i n a r y s t u d i e s , begun i n c o - o p e r a t i o n w i t h the F i s h e r i e s Research Board T e c h n o l o g i c a l Research L a b o r a t o r y , Vancouver, both immature Oncorhynchus  kisutch  (coho salmon) aged 6 t o 10  months, o b t a i n e d from the S t a t e of Washington Samish R i v e r , and Salmo  gaivdnevii  salmon h a t c h e r y ,  (steelhead t r o u t ) aged 6 t o  18 months o b t a i n e d from the S t a t e of Washington Game h a t c h e r y , Bellingham, were used. sequent s t u d i e s S. g a i v d n e v i i  Department of  In the m a j o r i t y of sub-  o b t a i n e d from January t o A p r i l  each year from the Bellingham hatchery were.kept the  Department  i n aquaria a t  of B i o c h e m i s t r y , U n i v e r s i t y o f B r i t i s h  Columbia.  These f i s h , aged 8 t o 10 months when o b t a i n e d , remained  sexually  immature and were s u i t a b l e f o r experiments over the succeeding 12 month p e r i o d . the  On o c c a s i o n Salmo  gaivdnevii  f r e s h water v a r i e t y of S. g a i v d n e v i i )  (rainbow t r o u t ,  aged 10 t o 18 months,  were o b t a i n e d from the Sun V a l l e y T r o u t Farm, Coquitlam, B.C. The f i s h were t r a n s p o r t e d by automobile from the h a t c h e r i e s to Vancouver  i n 20 g a l l o n c o n t a i n e r s .  The water which was  oxygenated throughout, c o n t a i n e d 1 t o 2 gm of a n a e s t h e t i c , MS 222 (Sandoz, or Kent Chemicals) and 2 gm Terramycin Animal formula) as a g e n e r a l p r o p h y l a c t i c measure.  (Pfizer,  Up t o 100  - 22 f i s h c o u l d be t r a n s p o r t e d i n t h i s manner i n each c o n t a i n e r without The  mortalities. fish,  i n groups of 20 t o 60, were kept i n 200 l i t r e  s e l f - c l e a n i n g f i b r e g l a s s a q u a r i a manufactured by H a l l c r a f t P l a s t i c s L t d . , Burnaby, B.C., or a l t e r n a t i v e l y , l a r g e r numbers were h e l d i n a 1200 l i t r e storage tank. nated by c h a r c o a l f i l t r a t i o n was sprayed  The water, d e c h l o r i -  ( J a c u z z i swimming p o o l  filter),  through s t a i n l e s s s t e e l a e r a t i o n n o z z l e s i n t o the  s m a l l a q u a r i a a t a r a t e of 2 t o 4 l i t r e s per minute, and i n t o the l a r g e r storage tank a t 10 to 12 l i t r e s per minute. water temperature i n the s m a l l a q u a r i a was maintained to  13°C by the use of a heat exchanger i n the supply  The a t 12°  line,  w h i l e the temperature i n the l a r g e tank v a r i e d from 6°C i n winter months t o 9 or 10°C i n the summer.  In most cases  temperature changes were made g r a d u a l l y over s e v e r a l weeks t o a l l o w the f i s h t o a c c l i m a t e .  The a q u a r i a were p r o v i d e d  adequate a e r a t i o n and a standard, photoperiod.  12 hour l i g h t ,  12 hour dark,  The f i s h were f e d p r o d u c t i o n s i z e p e l l e t  (J.R. C l a r k Co., S a l t Lake C i t y ,  with  food  Utah).  (b) C o l l e c t i o n of P i t u i t a r y Glands and P r e p a r a t i o n of P i t u i tary Extracts The tshawytscha  initial  supply of p i t u i t a r y glands  (chinook  from  Oncorhynchus  salmon), a g i f t of the F i s h e r i e s Research  Board, had been c o l l e c t e d a t the Green R i v e r Salmon hatchery, Washington i n 1963.  P i t u i t a r y glands  the spawning o p e r a t i o n s  were c o l l e c t e d  during  i n 1965 from 0. k i s u t c h a t Green R i v e r ,  - 23 and i n 1966 from 0. k i s u t e h  a t Green R i v e r and 0.  tshawytseha  a t the U n i v e r s i t y of Washington, F i s h e r i e s Research  Institute  h a t c h e r y , as w e l l as the S t a t e of Washington salmon hatchery a t Samish R i v e r .  In each case, cores from the b r a i n r e g i o n of the  salmon were o b t a i n e d w i t h an e l e c t r i c a l l y powered s t a i n l e s s steel cutting tool  (117).  The p i t u i t a r y glands, d i s s e c t e d  e a s i l y from these cores were f r o z e n over dry i c e and s t o r e d a t -20°C. P i t u i t a r y e x t r a c t s v/ere prepared e s s e n t i a l l y as d e s c r i b e d by Schmidt et al (118).  Frozen glands were homogenized a t 0°C  i n a Waring Blender w i t h two volumes of i s o t o n i c sodium c h l o r i d e f o r 3 minutes.  (0.9%,  The homogenate was c e n t r i f u g e d  (12,000 g, 30 min) a t 4°C and the supernatant l i q u i d  stored i n  5 ml a l i q u o t s a t -20°C u n t i l r e q u i r e d f o r i n j e c t i o n . e x t r a c t appeared  w/v)  The  s t a b l e t o l i m i t e d f r e e z i n g and thawing, and  to prolonged s t o r a g e over s e v e r a l months a t -20°C. (c) I n d u c t i o n of Spermatogenesis  i n Immature Salmonoid  Immature f i s h r e c e i v e d twice weekly i n j e c t i o n s  Fish  (0.05 ml)  of the salmon p i t u i t a r y e x t r a c t c o n t a i n i n g gonadotrophic a c t i v i t y f o r a p e r i o d of 8 t o 10 weeks.  P r i o r t o each  the f i s h were a n a e s t h e t i z e d w i t h 300 mg MS 222 i n f i v e of water.  In the i n i t i a l  and 100 S. g a i r d n e v i i of i n j e c t i o n s .  study 100 0. k i s u t e h  injection litres  (coho salmon)  ( s t e e l h e a d t r o u t ) were g i v e n the s e r i e s  F i s h were k i l l e d a t a p p r o p r i a t e i n t e r v a l s  over  the two month p e r i o d , the t e s t e s e x c i s e d and weighed, and a c i d e x t r a c t s of these t e s t e s prepared by b r i e f  homogenization  - 24 ( S o r v a l Omnimix) of the t i s s u e i n 5 volumes 0.2 M h y d r o c h l o r i c a c i d and c e n t r i f u g a t i o n (12,000 g, 30 min) a t 4°C.  The p r o t e i n s  i n the l y o p h i l i z e d e x t r a c t s were then examined by e l e c t r o p h o r e s i s i n polyacrylamide testis  gels.  F o r the subsequent s t u d i e s  c e l l suspensions a c t i v e i n the s y n t h e s i s  t e s t e s were e x c i s e d spermatogenesis.  utilizing  of protamine,  from the f i s h a t a l a t e stage of induced  T h i s was normally a f t e r 6 t o 8 weeks of the  twice weekly i n j e c t i o n s .  I t was u s u a l l y p o s s i b l e t o a s c e r t a i n  the sex and stage of m a t u r a t i o n of the f i s h by v i s u a l examinat i o n f o r with a large increase of the f i s h become q u i t e (d)  i n testis  weights the abdomen  distended.  Histology Samples of the t e s t e s were f i x e d i n Bouin's s o l u t i o n  (119)  or b u f f e r e d f o r m a l i n s o l u t i o n (120), and the author i s  g r a t e f u l t o Mr. C.F.A. C u l l i n g , Department of Pathology, U n i v e r s i t y of B r i t i s h  Columbia, f o r the s e c t i o n i n g and  s t a i n i n g o f these samples w i t h hematoxylin and e o s i n . (e) C h a r a c t e r i z a t i o n of the B a s i c P r o t e i n s by G e l E l e c t r o phoresis Throughout t h i s work the b a s i c p r o t e i n s from the t e s t e s c e l l s were r o u t i n e l y c h a r a c t e r i z e d by e l e c t r o p h o r e s i s i n p o l y acrylamide g e l s . gel  trays  (121).  These* g e l s were prepared i n standard  (O. H i l l e r  Co.) e s s e n t i a l l y as d e s c r i b e d  starch  by C r u f t  F i v e hundred ml of b u f f e r , normally 0.1 M sodium  a c e t a t e , pH 4.5, c o n t a i n i n g  50 gm acrylamide  (Matheson,  man and B e l l ) and 1 gm N,N-methylene b i s a c r y l a m i d e  Cole-  (Eastman)  - 25 was  degassed by evacuation  i n a s u c t i o n f l a s k , and  d i t i o n of c a t a l y s t s o l u t i o n s , 5.0  ml water, 0.5  -  (0.5 gm  after  ad-  ammonium p e r s u l p h a t e  in  ml N,N,N , N'-tetramethylethylenediamine 1  (Eastman) d i l u t e d to 50 ml w i t h methanol) poured i n t o the tray.  The  l i d w i t h t e f l o n s l o t formers was  the t r a y , the edges of which had  then p l a c e d  After polymerization had  been p r e c o o l e d  minimize h e a t i n g  the samples were a p p l i e d to the g e l which  t o 4°C.  The  e l e c t r o p h o r e s i s , a t 4°C  w i t h i n the g e l ) , was  s t a r c h g e l s by Smithies  (122).  The  (100  Depending on the c u r r e n t ,  to 200  v o l t s across  voltage Heathkit to 12 hours.  end  chambers  for  contained  (25 to 70 ma)  (w/v)  sodium  and  voltage  the 32 cm g e l ) , s u p p l i e d by a  constant  IP-32 power pack, e l e c t r o p h o r e s i s was  The  (to  c a r r i e d out i n a v e r t i -  M sodium c h l o r i d e , the e l e c t r o d e chamber 10%  chloride.  petro-  blocks.  c a l e l e c t r o p h o r e s i s apparatus, i n the manner d e s c r i b e d  0.15  over  been w e l l greased w i t h  latum to o b t a i n a good s e a l , and weighted w i t h l e a d  gel  for 4  g e l s , removed from the t r a y s a f t e r e l e c t r o -  p h o r e s i s , were s t a i n e d f o r 1 to 12 hours w i t h a s o l u t i o n of 1%  (w/v)  Amido Black 10B  then d e s t a i n e d by 0. H i l l e r  5 cm  acetic acid  and  supplied  Co. a l s o c a r r i e d out  in  polyacrylamide  long, of s i m i l a r composition to those  above, c a s t i n g l a s s tubing  (0.45  p l i f i e d d i s c g e l method, 0.01 10%  (v/v)  e l e c t r o p h o r e t i c a l l y i n an apparatus  E l e c t r o p h o r e s i s was gels  (Merck) i n 2%  sucrose was  x 8.0  (123)  In t h i s  sim-  ml of the p r o t e i n s o l u t i o n i n  l a y e r e d on the g e l , and  assembled i n an apparatus  cm).  described  the tubes then  f o r e l e c t r o p h o r e s i s over  40  - 26 to 60 minutes.  The  g e l s , removed from the t u b i n g by  gently  rimming w i t h a d i s s e c t i n g needle were s t a i n e d f o r 30 minutes w i t h a 0.1 i n 10% (f)  % (w/v)  s o l u t i o n of Amido Black  10B  and  then p l a c e d  (v/v) a c e t i c a c i d f o r d e s t a i n i n g .  B i o s y n t h e s i s of Protamine i n T e s t i s C e l l (i) I n c u b a t i o n  Suspensions  procedures:  . T e s t i s c e l l s a t a stage a c t i v e i n protamine b i o s y n t h e s i s were obtained  from S. g a i v d n e r i i  t o g e n e s i s .had been induced e x t r a c t s as d e s c r i b e d t i v e source  i n which sperma-  by the i n j e c t i o n s of salmon' p i t u i t a r y  i n a preceding  section (c).  of t e s t i s c e l l s a t a s i m i l a r stage was  t e s t e s of 0. nerka salmon).  (steelhead)  An a l t e r n a the maturing  (sockeye salmon) or 0. tshawytscha  (chinook  Both s p e c i e s of salmon c o u l d be caught d u r i n g  spawning m i g r a t i o n s August 1965  upriver.  0. nerka  were obtained  in  by d i p n e t t i n g immediately below'the Bridge  r a p i d s i n the F r a s e r R i v e r , B.C.,  w h i l e 0. tschawytscha  their midRiver were  n e t t e d i n the shallow water of the Samish R i v e r , Washington i n l a t e September 1965. the author i s indebted  For a s s i s t a n c e i n o b t a i n i n g these to the I n t e r n a t i o n a l P a c i f i c  F i s h e r i e s Commission, the Indians v a t i o n , and  fish  Salmon  of the B r i d g e R i v e r r e s e r -  the S t a t e of Washington Department of F i s h e r i e s .  T e s t i s t i s s u e from the salmon.was b r i e f l y minced with s c i s s o r s and  suspended i n Hanks medium (Baltimore B i o l o g i c a l  a t 0°C  f o r t r a n s p o r t a t i o n to Vancouver.  Incubation  Laboratory) experi-  ments were begun 1 t o 6 hours a f t e r removal of the t e s t e s from the  fish.  -  27  -  Suspensions of i n t a c t c e l l s were prepared from t e s t e s of these f i s h .  One ml a l i q u o t s of s c i s s o r - m i n c e d  t e s t e s were  g e n t l y homogenized i n 5 ml Hanks medium a t 0°C i n P o t t e r Elvehjem tubes w i t h a T e f l o n p e s t l e . was twice c e n t r i f u g e d medium.  The r e s u l t i n g suspension  (900 g, 5 min) and resuspended i n Hanks  The c e l l s a t a f i n a l d i l u t i o n of 1 ml packed c e l l s i n  8 ml Hanks medium were preincubated  i n 1.0 ml a l i q u o t s a t 0°C  for  i n h i b i t o r , puromycin (as  90 minutes w i t h the a p p r o p r i a t e  the d i h y d r o c h l o r i d e , heximide  N u t r i t i o n a l Biochemical Corp.), c y c l o -  ( N u t r i t i o n a l B i o c h e m i c a l Corp.), chloramphenical (a  g i f t of Dr. W.E.  Razzell)  Sharpe and Dohme). 5 uC (microcuries) with L - a r g i n i n e - C  1 2  and a c t i n o m y c i n D (a g i f t of Merck,  The i n c u b a t i o n s L-arginine-H  3  were begun by a d d i t i o n of  (Volk,  1.4 C/mMole)  .to g i v e a f i n a l c o n c e n t r a t i o n  (diluted  of a r g i n i n e  -5 of 1 x 10  M) and t r a n s f e r of the tubes t o a 20°C water bath  with a gyratory  shaker.  The i n c u b a t i o n s  were stopped a t the  s p e c i f i e d times by the a d d i t i o n of 2.0 ml Hanks medium made 1 x 10 0°C.  M i n u n l a b e l l e d a r g i n i n e and immediately c o o l e d t o  A f t e r c e n t r i f u g a t i o n (900 g, 5 min) the packed  cells  were suspended i n 0.5 ml water and f r o z e n . ( i i ) P u r i f i c a t i o n of r a d i o a c t i v e protamine: Protamine was p u r i f i e d by e l e c t r o p h o r e s i s of the 0.2 M hydrochloric The  a c i d e x t r a c t s from the c e l l s  f r o z e n c e l l s from the i n c u b a t i o n s  i n polyacrylamide  gels.  were thawed, t r a n s f e r r e d  to g l a s s P o t t e r - E l v j h e m  tubes w i t h 2 ml of 0.2 M h y d r o c h l o r i c  a c i d f o r homogenization  (2500 rpm, 1 min) w i t h a g l a s s - T e f l o n  pestle  ( T r i - R homogenizer). " The homogenates were c e n t r i f u g e d  - 28 (12,000 g, 60 min)  and the supernatant  The r e s i d u e s were then d i s s o l v e d i n 0.2 c l a r i f i e d by c e n t r i f u g a t i o n natants  (volume about 0.19  acrylamide g e l .  The  l a r g e r dimensions  extracts lyophilized. ml water, the s o l u t i o n s  (12,000 g, 30 min) ml)  and the  super-  p l a c e d i n the s l o t s of a p o l y -  sample s l o t s , f o u r per g e l , were made to  (0.15 x 1.9  cm)  to accomodate t h i s volume.  The g e l s were then run as p r e v i o u s l y d e s c r i b e d (200 v o l t s , 4 hours) a t 4°C. was a 1%  determined (w/v)  The p o s i t i o n of the protamine  band i n the g e l  by s u r f a c e s t a i n i n g the g e l f o r one minute w i t h  aqueous s o l u t i o n of Wool F a s t Blue BL  (0. H i l l e r  Co.), a dye which p e n e t r a t e s the p o l y a c r y l a m i d e g e l slowly.  The s t a i n e d protamine  area was  extremely  then c u t out, minced  by p a s s i n g through a d i s c of s t a i n l e s s s t e e l s c r e e n i n g  (150  mesh) i n a 2.5 ml p l a s t i c s y r i n g e , and the g e l p a r t i c l e s packed i n a s m a l l column  (0.8 cm diameter)  0.1 M a c e t i c a c i d .  The  f o r e l u t i o n w i t h 10  ml  l y o p h i l i z e d eluates containing prota-  mine were h y d r o l y z e d w i t h 5.7  M h y d r o c h l o r i c a c i d a t 105°C f o r  16 hours, d r i e d under vacuum and d i s s o l v e d i n water.  Suitable  a l i q u o t s were removed f o r c o u n t i n g i n 10 ml of Bray's  scintil-  lation fluid  (124)  u s i n g a Nuclear Chicago  f o r a r g i n i n e d e t e r m i n a t i o n by a Sakaguchi f i e d t o i n c l u d e a p r e a c e t y l a t i o n step i n water was hydroxide  and  c o o l e d to 0°C and 0.15  was  (w/v)  added.  2%  and  (125), modi-  A one ml  aliquot  potassium A f t e r standing  2.0 ml of 8-hydroxyquinoline  8-hydroxyquinoline,  sodium hydroxide)  reaction  (126).  ml 10%  ml a c e t i c anhydride  f o r one hour a t 0°C, (0.02% (w/v)  2.0  U n i l u x counter,  reagent  (v/v) methanol i n 3 N  added, the tube shaken and  1.0  ml  0.1%  - 29 (w/v) M bromosuccinimide i n water added. 500 mu was  The absorbance a t  r e a d a f t e r 10 minutes a t room temperature i n a  Beckman DB spectrophotometer a g a i n s t the reagent blank. (g) P u r i f i c a t i o n and C h a r a c t e r i z a t i o n of Protamine T e s t e s from S. g a i v d n e r i i , as w e l l as c e r t a i n of the P a c i f i c salmon, f r o z e n a f t e r removal from the f i s h were used as s t a r t i n g m a t e r i a l i n s e v e r a l s t u d i e s on the p u r i f i c a t i o n and c h a r a c t e r i z a t i o n on protamine.  In l a t e r s t u d i e s of the  amino a c i d s at the N and C - t e r m i n a l s of protamine from S. g a i v d n e v i i , mature spermatozoa were c o l l e c t e d by e x p r e s s i n g m i l t from the t r o u t d u r i n g spawning hatchery on the S k a g i t R i v e r of  Game).  operations at a f i s h  (State of Washington,  Department  Mature spermatozoa were s i m i l a r l y c o l l e c t e d  a d u l t 0. k i s u t c h Skagit River  from  (coho salmon) a t a second hatchery on the  (State of Washington,  Department  of F i s h e r i e s ) .  E i t h e r f r o z e n t e s t i s t i s s u e or spermatozoa o b t a i n e d after centrifugation  (10,000 g, 5 min)  e x t r a c t e d w i t h 2 t o 3 volumes  of m i l t , were twice  of 0.2 M h y d r o c h l o r i c a c i d by  homogenization a t 0°C i n a S o r v a l Omnimix. gation  (12,000 g, 30 min), the combined  were l y o p h i l i z e d .  The d r i e d m a t e r i a l was  (Pharmacia) . nal  on Sephadex G-25,  then d i s s o l v e d i n (500 g, 10  f i n e beaded  The column, normally 40 x 2.0 cm  d i a m e t e r ) , was  centrifu-  supernatant e x t r a c t s  water, c l a r i f i e d by a b r i e f c e n t r i f u g a t i o n and chromatographed  After  min)  form  (length x i n t e r -  e l u t e d w i t h 0.1 M a c e t i c a c i d , the e f f l u e n t  c o l l e c t e d i n 5 ml f r a c t i o n s , and p r o t e i n c o n c e n t r a t i o n i n each  '-' 30  -  f r a c t i o n measured i n a Beckman DB ing the absorbance a t 220,  230  spectrophotometer by  or 280  my  as a p p r o p r i a t e .  p r o t e i n peaks were pooled, l y o p h i l i z e d and s m a l l volume (0.5 P-10  f i e d by  larly  Two  column (2.0  M acetic acid.  x 30 cm)  P r o t e i n peaks, again  or L - s e r i n e - C  a c i d e x t r a c t was  Laboratories)  1 k  i n t o protamine  column i n the a c e t a t e form and  e l u t i n g w i t h 0.1  of  Biogel was  identi-  particu-  inorganic  (see p a r t I I ) ;  passed through a Dowex 1 x 8  a l s o t h i s e x t r a c t was  a  l y o p h i l i z e d as  i n experiments i n v o l v i n g i n c o r p o r a t i o n 32  (Bio-Rad  e l u t e d w i t h water,  chromatographed on Sephadex  M acetic acid.  The  The in a  which  a d d i t i o n a l steps were o f t e n i n c l u d e d ,  phosphate-P  G-10  p r o t e i n peak emerging  immediately a f t e r the v o i d volume was on  redissolved  f o r rechromatography on  spectrophotometry, were pooled and  before.  and  ml)  (Bio-Rad L a b o r a t o r i e s )  e l u t e d w i t h 0.2  the  to 2.0  record-  used i n f u r t h e r  studies  protamine. The  e x t e n t of p u r i f i c a t i o n was  electrophoresis  e a s i l y monitored  of s u i t a b l e a l i q u o t s from the v a r i o u s  i n p o l y a c r y l a m i d e g e l s as d e s c r i b e d  by stages  i n a preceding s e c t i o n  Samples f o r amino a c i d a n a l y s i s were h y d r o l y z e d i n 6.0 hydrochloric time and analyzer.  a c i d i n vacuo  at 105°C f o r v a r i o u s  amino a c i d  N - t e r m i n a l amino a c i d s were i d e n t i f i e d by  reac-  t i o n w i t h l-dimethylaminonaphthalene-5-sulfony1 c h l o r i d e ( A l d r i c h Chemical Co.)  a c c o r d i n g to Gray and  followed  i n 6.0 M h y d r o c h l o r i c  by h y d r o l y s i s  hours, u n l e s s p r o l i n e was  Hartley acid  M  p e r i o d s of  the h y d r o l y s a t e s analyzed on a Beckman 120C The  (e).  (127)  (105°C,  expected i n which case a 6 hour  16  - 31 h y d r o l y s i s time was used) and subsequent i d e n t i f i c a t i o n of the DNS-amino a c i d s by t h i n l a y e r chromatography on s i l i c a g e l G  by Dr. J.A. B l a c k  (Merck) u s i n g the s o l v e n t systems developed  (128)  ( s o l v e n t system A:  chloroform/methanol/acetic  90/10/1 by volume, and s o l v e n t system B: hydroxide,  80/20).  ymoles of protamine distilled)  n-propanol/ammonium  A l t e r n a t i v e l y , end groups were  by use of a s u b t r a c t i v e Edman technique  acid,  (129).  determined  One t o 5.0  were d i s s o l v e d i n 0.4 ml 50% p y r i d i n e  and the Edman d e g r a d a t i o n performed  (re-  i n a 3 ml t e s t  tube by a d d i t i o n of 0.0 50 ml N-methylmorpholine and 0.020 ml phenylisothiocyanate.  The contents of the tube were mixed  and allowed t o r e a c t f o r two hours a t 37°C.  A f t e r d r y i n g down  i n a vacuum d e s s i c a t o r , 0.2 ml of t r i f l u o r o a c e t i c a c i d was added and c y c l i z a t i o n allowed t o proceed for  a t room  temperature  one hour b e f o r e d r y i n g down a g a i n a t 60°C i n a vacuum  d e s s i c a t o r over sodium hydroxide.  Water, 0.2 ml was added and  the s o l u t i o n e x t r a c t e d t h r e e times w i t h 1.0 ml n - b u t y l a c e t a t e , d r i e d down and the sample taken up i n 50% p y r i d i n e .  An a l i -  quot was then removed f o r a c i d h y d r o l y s i s and subsequent amino a c i d a n a l y s i s .  In the C - t e r m i n a l amino a c i d  0.1 ml a l i q u o t s o f a s o l u t i o n of S. g a i v d n e v i i  determination.  protamine  (10  mg/ml) i n 0.1 M ammonium b i c a r b o n a t e were incubated a t 37°C for  one hour w i t h 0.005 ml of e i t h e r carboxypeptidase  pension ton) .  (Worthington)  or carboxypeptidase  B solution  A sus(Worthing-  The d i g e s t s were s p o t t e d on Whatman 3 MM paper along -  w i t h s u i t a b l e amino a c i d markers and h i g h v o l t a g e e l e c t r o p h o r e s i s was c a r r i e d out a t pH 6.5 ( p y r i d i n e / a c e t i c  acid/water,  - 32  -  100/4/900 by volume) i n a V a r s o l - c o o l e d minutes a t 4000 v o l t s and  (62 v/cm).  stained with ninhydrin  acetone, 2 p a r t s  1.0  The  (8 p a r t s  M cadmium  apparatus f o r  paper was  1%  (w/v)  20  then d r i e d  ninhydrin  in  acetate).  (h) F r a c t i o n a t i o n of Protamine (i) Chromatography on carboxymethyl Sephadex: Protamine from 0.  tshawytscha  t e s t e s , p u r i f i e d by  two  step chromatography procedure d e s c r i b e d  was  used i n these s t u d i e s .  Ten  chromatographed on a column Sephadex  11.4,  and  (40 x 1.0 cm)  mixing chamber and  2 00 ml  buffer reservoir.  F i v e ml  fraction.  200  of 2.0  ml  The  estimated by a Sakaguchi ml  a l i q u o t s of each  c o n d u c t i v i t y of the e l u a t e was (type - CDM  2d).  tshawytscha  (25 x 1.0  Oxide, Woelm) e q u i l i b r a t e d and hydrogen phosphate.  E i g h t ml  cm)  protamine of alumina  e l u t e d w i t h 0.45  was  (Aluminium  M dipotassium  f r a c t i o n s were c o l l e c t e d and  protamine c o n c e n t r a t i o n  estimated by  peak was  as i n d i c a t e d i n F i g u r e  d i v i d e d i n two  measured with a  alumina:  of the p u r i f i e d 0.  chromatographed on a column  the  M sodium c h l o r i d e i n the same  a p p l i e d d i r e c t l y to 0.2  ( i i ) Chromatography on  of sodium  f r a c t i o n s of the e l u a t e were c o l -  Radiometer c o n d u c t i v i t y meter  Twenty mg  M sodium carbonate  of t h i s b u f f e r i n  the protamine c o n c e n t r a t i o n  (125)  was  of carboxymethyl  e l u t e d with a : l i n e a r g r a d i e n t  c h l o r i d e e s t a b l i s h e d by using  l e c t e d and  i n (g) above,  of t h i s protamine  (Pharmacia), e q u i l i b r a t e d w i t h 0.1  b u f f e r , pH  reaction  mg  the  absorbance at 220 11 and  my. each  the The  - 33 p o r t i o n A and B, rechromatographed on the same column a f t e r desalting.  T h i s d e s a l t i n g of the s e p a r a t e l y pooled f r a c t i o n s  A and B was accomplished by a p p l y i n g each t o a column cm) of carboxymethyl Sephadex and e l u t i n g w i t h h y d r o x i d e , 1.0 M formic a c i d and f i n a l l y acid.  (6 x 1.5  1.0 M ammonium  1.0 M h y d r o c h l o r i c  The 1.0 M h y d r o c h l o r i c a c i d e l u a t e c o n t a i n i n g the pro-  tamine was l y o p h i l i z e d and r e d i s s o l v e d i n 0.5 ml 0.45 M i d i potassium hydrogen phosphate f o r the rechromatography on alumina. RESULTS and DISCUSSION (a) I n d u c t i o n  of Spermatogenesis i n Salmonoid F i s h  That protamine occurs spermatogenesis was f i r s t  o n l y a t a r e l a t i v e l y l a t e stage of noted by Miescher  s t u d i e s of the Rhine salmon, Salmo  salar.  (1,4) i n h i s The t e s t e s o f these  f i s h , weighing only s e v e r a l grams when the f i s h entered water, i n c r e a s e d shortly before  fresh  i n s i z e and c o u l d weigh s e v e r a l hundred grams  spawning.  ture, considerably  Miescher concluded t h a t the muscula-  reduced d u r i n g  t h i s p e r i o d , was used f o r  the growth of the gonads, i n p a r t i c u l a r f o r the formation of protamine. F e l i x et al (24) c o l l e c t e d Salmo  fontinalis  (brook t r o u t )  t e s t e s a t i n t e r v a l s p r i o r t o spawning and was a b l e t o show the presence of protamine i n t e s t e s obtained spawning but not i n e a r l i e r samples. reached i n the h i s t o c h e m i c a l  about 40 days  A s i m i l a r c o n c l u s i o n was  s t u d i e s of A l f e r t  maturing t e s t e s of 0. tshawytsaha.  before  (26) on the  By means of the d i f f e r e n t  - 34 s o l u b i l i t i e s of h i s t o n e s and protamine  in trichloroacetic  and t h e i r r e a c t i o n w i t h the s t a i n "Fast Green" he  demonstrated  t h a t the n u c l e a r p r o t e i n i s suddenly transformed a t a l a t e stage of spermatogenesis  acid  fairly  from the h i s t o n e type s i m i l a r t o  t h a t found i n somatic t i s s u e s to the protamine  type.  These  changes took p l a c e w e l l a f t e r completion of m e i o s i s .  Simi-  l a r l y an examination by Ando and Hashimoto (25) of the b a s i c p r o t e i n s p r e s e n t i n the t e s t e s of Salmo t r o u t ) a t monthly i n t e r v a l s throughout the f o r m a t i o n of protamine to the m a t u r a t i o n of  gaivdnevii  (rainbow  the year showed t h a t  i n the t e s t i s n u c l e i ran p a r a l l e l  spermatozoa.  Thus i n any b i o c h e m i c a l study of protamine  a supply of  t e s t e s t i s s u e a t the a p p r o p r i a t e stage of development becomes a c r i t i c a l f a c t o r determining the nature of experiments can be undertaken.  In t h a t the s p e c i e s of salmonoid  a v a i l a b l e l o c a l l y mature o n l y a t one  that  fish  season of the year, and  hence are a c t i v e i n the s y n t h e s i s of protamine  at only t h i s  one season, s t u d i e s r e q u i r i n g v i a b l e c e l l s might have been r e s t r i c t e d t o a r a t h e r f r a n t i c one or two month p e r i o d . u t i l i z i n g a number of the d i f f e r e n t s p e c i e s of P a c i f i c or t r o u t , these s t u d i e s c o u l d have been extended  By salmon  over t h r e e or  four months, f o r these d i f f e r e n t s p e c i e s do not mature s e x u a l l y a t the same time of year. have a year-round  O b v i o u s l y i t would be d e s i r a b l e to  source of t e s t e s t i s s u e a t the a p p r o p r i a t e  stage of m a t u r a t i o n . The presence i n t e l e o s t f i s h of a. p i t u i t a r y  gonadotrophin  w i t h p h y s i o l o g i c a l and b i o c h e m i c a l c h a r a c t e r i s t i c s very  similar  - 35 to those of the mammalian l u t e i n i z i n g hormone, LH, has established  (37).  A p a r t i a l l y p u r i f i e d gonadotrophic p r e -  p a r a t i o n from the l y o p h i l i z e d p i t u i t a r i e s of spawning (0. keta)  been  salmon  produced marked enlargement of the gonads and sper-  matogenesis when i n j e c t e d or implanted i n immature S. nevii  (130) .  gaivd-  In a f u r t h e r a p p l i c a t i o n of t h i s work (118) ,  the i n c r e a s e i n t e s t i s weights r e s u l t i n g  from a s e r i e s of  s i x i n j e c t i o n s of p i t u i t a r y e x t r a c t over a two week p e r i o d was  the b a s i s of a b i o a s s a y f o r gonadotrophic a c t i v i t y . In the p r e s e n t s t u d i e s , the response of immature  hynchus  kisuteh  and  Salmo  gaivdnevii  (steelhead  Oncov-  trout)  to a  s e r i e s of twice weekly i n j e c t i o n s over a two month p e r i o d of the p i t u i t a r y e x t r a c t was  examined  initially.  The weights of  the t e s t e s of both s p e c i e s of f i s h r e c e i v i n g the p i t u i t a r y  ex-  t r a c t were o b t a i n e d a t a p p r o p r i a t e i n t e r v a l s throughout the injection period.  The average t e s t i s weight r e c o r d e d a t each  of these i n t e r v a l s i s shown i n F i g u r e 2(a) f o r 0. k i s u t e h F i g u r e 2(b) f o r S.  gaivdnevii.  As r e p o r t e d by Robertson and R i n f r e t et al  and  (130) , and  Schmidt  (118), the weights of the t e s t e s i n immature salmonoid  f i s h r e c e i v i n g salmon p i t u i t a r y e x t r a c t s i n c r e a s e d remarkably. Thus, f o r s t e e l h e a d t r o u t the t e s t i s weights i n immature  fish,  from 10 t o 20 mg at the s t a r t of i n j e c t i o n s , were seen to i n c r e a s e to w e l l over 1.0 gm.  I t should be noted t h a t w h i l e  the average t e s t i s weight has been p l o t t e d , there was, c u l a r l y a t the l a t e r stages of development,  parti-  a r a t h e r wide  d i s t r i b u t i o n of weights f o r any one o b s e r v a t i o n p e r i o d .  For  -  36  -  (A) 0 . k i s u t c h 300  E sz cn  (B)  oO  S.gairdneri  CD CD CD <TJ  I— •  1500  > <  1000  500 •  0  0  14  28  . Days After Start of  42  56  Injections  F i g u r e 2 - Average t e s t i s weights o f 0. (A)  and S. g a i r d n e r i i  kisutch  (B) e x c i s e d a t i n t e r v a l s  a f t e r the s t a r t o f i n j e c t i o n s from p i t u i t a r y glands, o f 0...  o f an extract^ (118) tshawytscha.  - 37 example, a t 49 days a f t e r  the s t a r t of the i n j e c t i o n  the t e s t i s weights ranged from 90 to 370 mg and  from 1.06  to 2.90  gm  increasingly d i f f i c u l t  f o r 0.  f o r S. g a i v d n e v i i .  series  kisuteh,  I t a l s o became  to o b t a i n an a c c u r a t e measure of  t e s t i s weights a t these l a t e r stages  the  of development f o r a  c o n s i d e r a b l e p r o p o r t i o n of the t e s t e s a t t h i s stage was ure sperm which was fish.  l o s t on removal of the t e s t i s from  Schmidt et al  (118)  of the gonads t o p i t u i t a r y of the f i s h . l e n g t h 14.9  The + 1.4  have noted t h a t the  f  and  e x t r a c t i n c r e a s e d with  body weight 28.4  the  sensitivity  response of the S. g a i v d n e v i i cm  mat-  the  length  (average  ± 12.7  gm)  was,  not unexpectedly, c o n s i d e r a b l y g r e a t e r than t h a t f o r the kisuteh 8.1  (average l e n g t h 13.4  ± 1.2  cm  and  0.  body weight 23.4  ±  gm). Extending the i n j e c t i o n s of t h i s e x t r a c t from  glands of a d u l t salmon over a p e r i o d of two shown i n the present  study to r e s u l t  t e s t e s of immature salmonoid f i s h .  pituitary  months has  been  i n the m a t u r a t i o n of As  i t was  c o l l e c t l a r g e numbers of p i t u i t a r y glands  p o s s i b l e to  (over 9000 i n  from a d u l t salmon d u r i n g the annual spawning o p e r a t i o n s f i s h hatcheries this pituitary  the  1966) at  i n Washington S t a t e , s u f f i c i e n t q u a n t i t i e s of e x t r a c t have been a v a i l a b l e to m a i n t a i n  stocks  of young t r o u t i n a q u a r i a at the stage of m a t u r a t i o n s u i t a b l e f o r s t u d i e s on  biosynthesis.  The marked i n c r e a s e i n weight of the t e s t e s over  shown as the average + standard .^deviation  (131)  the  -  p e r i o d of the  -  38  i n j e c t i o n s of p i t u i t a r y e x t r a c t i s accompanied  by both b i o c h e m i c a l  and  h i s t o l o g i c a l changes i n the  To c h a r a c t e r i z e the b a s i c p r o t e i n s present at various  stages d u r i n g  i n the t e s t i s  0.2  of t e s t i s t i s s u e on p o l y a c r y l a m i d e  g e l s was  found to r e s u l t i n e x t e n s i v e  ferences  t h i s t a i l i n g can  em-  M hydrochloric acid extracts used.  t r o p h o r e s i s of these b a s i c p r o t e i n s i n s t a r c h or  bands and  cells  t h i s induced m a t u r a t i o n a system  p l o y i n g e l e c t r o p h o r e s i s of the  g e l s was  testes.  The  elec-  starch-urea  t a i l i n g of the  protein  e a s i l y o b l i t e r a t e the s u b t l e  i n the m o b i l i t y of p r o t e i n s  i n these media.  dif-  Poly-  a c r y l a m i d e , with i t s s u p e r i o r r e s o l u t i o n of p r o t e i n s and  a  minimum of problems r e s u l t i n g from the i n t e r a c t i o n of p r o t e i n and  g e l media, has  c a l and  been e x p l o i t e d r e c e n t l y f o r both  preparative  gel electrophoresis  (132,133).  analytiA  simple  technique employing the same apparatus as used i n s t a r c h g e l e l e c t r o p h o r e s i s had  been d e s c r i b e d  (121), and  polyacrylamide  g e l s prepared i n t h i s manner were used i n most of the  studies  c h a r a c t e r i z i n g the b a s i c p r o t e i n s of t e s t i s t i s s u e . The  t e s t e s of S. g a i r d n e r i i  over the course of the the p r o t e i n s present  i n the  0.2  M hydrochloric  gel electrophoresis.  acid The  and  extracts appear-  p r o t e i n band, 52 days a f t e r the s t a r t of i n -  j e c t i o n s , migrating seen i n F i g u r e  at i n t e r v a l s  i n j e c t i o n s of p i t u i t a r y e x t r a c t  examined by p o l y a c r y l a m i d e ance of a new  were e x c i s e d  3.  more r a p i d l y toward the cathode can  be  T h i s band, which corresponded i n m o b i l i t y  to protamine e x t r a c t e d  from n a t u r a l l y maturing t e s t e s t i s s u e  or spermatozoa, v/as not present  a t the e a r l i e r  stages of  the  - 39 -  PROTAMINE  jHISTONES  DAYS Figure  27  34  44  52  3 - Polyacrylamide g e l electrophoresis  the p r o t e i n s  extracted  testes excised  by 0.2 M HC1  gairdnerii  The a c i d e x t r a c t s were a p p l i e d  s l o t s a t the o r i g i n and a f t e r  f o r 4 hours a t 200 v o l t s , the g e l was Black 10B.  of  a t the i n d i c a t e d i n t e r v a l s from the s t a r t of  p i t u i t a r y extract injections. i n the sample  from S.  a t pH 4.5  A new  electrophoresis  s t a i n e d w i t h Amido  p r o t e i n band, w i t h the m o b i l i t y of p r o t a -  mine, i s seen i n the e x t r a c t from t e s t e s 52 days a f t e r the s t a r t of the i n j e c t i o n s of p i t u i t a r y e x t r a c t .  - 40 induced t e s t e s m a t u r a t i o n . slower m i g r a t i n g  At these e a r l i e r stages o n l y  b a s i c p r o t e i n s were observed.  of the b a s i c p r o t e i n s p r e s e n t immature S. g a i v d n e v i i  Figure  4.  A comparison  i n an a c i d e x t r a c t of t e s t e s from  (steelhead)  which had  weeks of the p i t u i t a r y e x t r a c t and from S. g a i v d n e v i i  the  received  eight  the same e x t r a c t of  testes  (rainbow) maturing n a t u r a l l y i s shown i n  Both p r e p a r a t i o n s  were very  similar, differing  o n l y i n the r e l a t i v e i n t e n s i t i e s of each p r o t e i n band. The  development of the t e s t e s of immature t r o u t i n r e -  sponse to i n j e c t i o n s of p i t u i t a r y e x t r a c t s p a r a l l e l e d t h a t of the n a t u r a l l y maturing P a c i f i c t e s t e s contained mg had  salmon  (26).  o n l y c y s t s of spermatogonia a t the  weight range, y e t a f t e r 2 months complete occurred  The  t h i s induced m a t u r a t i o n or spermatogenesis i t was l a r g e numbers of spermatid c e l l s were present  detected The  86),  During not  until  i n the h i s t o l o be  i n the a c i d e x t r a c t of t e s t i s t i s s u e . replacement of one  fertilization,  These changes during f i r s t recognized  type of b a s i c n u c l e a r  and  spermatogenesis  by (26,134,  (92).  spermatogenesis i n f i s h were, of course,  i n the p i o n e e r i n g  demonstrated i n the h i s t o c h e m i c a l (26).  protein  e a r l y embryonic development  s t u d i e s of Miescher  K o s s e l on the maturing t e s t e s of S.  tshawytsoha  20  t h a t the appearance of protamine c o u l d  another i s a process which occurs d u r i n g 27,  10 to  differentiation  and mature spermatozoa were produced.  g i c a l preparations  immature  salav  (1,2,4), and  s t u d i e s of A l f e r t on  and were 0.  C h a r a c t e r i z a t i o n by g e l e l e c t r o p h o r e s i s  the b a s i c p r o t e i n s present  of  i n an a c i d e x t r a c t of t e s t i s t i s s u e  Protamine  Histones  F i g u r e 4 - Comparison by d i s c g e l e l e c t r o p h o r e s i s a t pH 4.5 of the p r o t e i n s e x t r a c t e d by 0.2 M from the testes of the immature S.  HC1  gaivdnevii  ( s t e e l h e a d t r o u t ) which had been r e c e i v i n g i n j e c t i o n s of the p i t u i t a r y e x t r a c t n a t u r a l l y maturing S.  (A), and the t e s t e s o f  gaivdnevii  (rainbow t r o u t ) (B).  - 42 d u r i n g hormonally induced  maturation  of immature S.  gaivdnevii  t e s t i s t i s s u e a l s o showed t h i s same marked change i n the complement of b a s i c n u c l e a r p r o t e i n s . when l a r g e numbers of spermatid  Protamine, only  detected  c e l l s were p r e s e n t ,  appeared  to r e p l a c e the h i s t o n e s at a l a t e stage of spermatogenesis. (b) The  B i o s y n t h e s i s of Protamine  The  s u i t a b i l i t y of salmonoid t e s t i s c e l l s f o r a  of p r o t e i n s y n t h e s i s has the s m a l l s i z e and  been emphasized a l r e a d y .  However  unusual amino a c i d composition  tamines a l s o r a i s e d the p o s s i b i l i t y t h a t these  study  of the  pro-  polypeptides  might be assembled by a mechanism s i m i l a r to t h a t e l u c i d a t e d f o r the s y n t h e s i s of the s h o r t b a c t e r i a l p o l y p e p t i d e s , cidin  (135)  and  g r a m i c i d i n S (136).  Evidence has  tyro-  accumulated  to show t h a t the b i o s y n t h e s i s of these p o l y p e p t i d e s  i s cata-  l y z e d with o n l y the s o l u b l e f r a c t i o n s obtained  strains  of B a c i l l u s  b v e v i s , and  t h a t t h i s s y n t h e s i s i s not a f f e c t e d by  compounds which are powerful on ribosomes.  The  i n h i b i t o r s of p r o t e i n s y n t h e s i s  i n i t i a l contradictory findings indicating  t h a t g r a m i c i d i n S was system  from  s y n t h e s i z e d by a mRNA, tRNA,  (137,138) have s i n c e been r e t r a c t e d (139)  ribosomal  and  the  char-  a c t e r i z a t i o n and p u r i f i c a t i o n of the enzyme systems i n v o l v e d i n s y n t h e s i s of t h i s p e p t i d e have been r e p o r t e d In c o n t r a s t to the s y n t h e s i s of these peptides,  the hormonal p o l y p e p t i d e s ,  (140,  141).  small b a c t e r i a l  poly-  insulin  (142)  c o r t i c o t r o p h i c hormone (ACTH) (143), appear to be by the r i b o s o m a l  system.  I t was  and  adreno-  synthesized  t h e r e f o r e of importance to  - 43 e s t a b l i s h the g e n e r a l nature of the protamine s y n t h e s i z i n g system  i n t e s t i s c e l l s b e f o r e any i n v i t r o  of the components of such a system was  characterization  initiated.  With a supply of t e s t i s t i s s u e a t a stage of m a t u r a t i o n s u i t a b l e f o r s t u d i e s of protamine  s y n t h e s i s a s s u r e d , i t was  necessary t o develop methods t o examine the i n c o r p o r a t i o n of r a d i o a c t i v e p r e c u r s o r amino a c i d s i n t o protamine.  Testis  cells  a t the a p p r o p r i a t e stage of m a t u r a t i o n c o u l d be o b t a i n e d from e i t h e r a d u l t salmonoid  f i s h undergoing  immature S. g a i r d n e r i i  i n which t h i s maturation had been i n -  duced hormonally  n a t u r a l m a t u r a t i o n or  by i n j e c t i o n s of the p i t u i t a r y  extract.  T e s t i s c e l l s were e a s i l y d i s s o c i a t e d by a g e n t l e hand homogeni z a t i o n technique  (144)  to g i v e a suspension of i n t a c t  and upon i n c u b a t i o n of such a c e l l  cells,  suspension a t 20°C i n buf-  f e r e d Hanks medium w i t h the r a d i o a c t i v e p r e c u r s o r , a r g i n i n e H, 3  i n c o r p o r a t i o n of a r g i n i n e - H  3  i n t o protamine was  The methods used f o r the assay of t h i s i n t o protamine  observed.  incorporation  m e r i t some d i s c u s s i o n a t t h i s p o i n t .  The  c l a s s i c a l methods f o r the p u r i f i c a t i o n of protamine  have  normally i n v o l v e d a c i d or high s a l t e x t r a c t i o n f o l l o w e d by p r e c i p i t a t i o n of protamine (145), acetone  by p i c r i c a c i d  (4) metaphosphate  (4), or t r i c h l o r o a c e t i c a c i d  (146).  A l l of  these methods however s u f f e r from a lack of s p e c i f i c i t y  and  although they were q u i t e s a t i s f a c t o r y f o r the i s o l a t i o n of protamine  from mature spermatozoa, the presence  stages of spermatogenesis  at e a r l i e r  of a v a r i e t y of c e l l types  31) c o n t a i n i n g b a s i c p r o t e i n s other than protamine t h e i r use i n t h i s system.  A method of protamine  (26,30,  precludes  purification  - 44  -  u t i l i z i n g g e l f i l t r a t i o n on Sephadex G-25 be d e s c r i b e d  i n a following section  protamine f r e e of any processing  to  contaminants does not permit the r a p i d  of l a r g e numbers of i n d i v i d u a l ' i n c u b a t i o n s .  t i o n of protamine i n such c e l l  f o r the  For  purifica-  suspension s t u d i e s .  c l e a r r e s o l u t i o n of protamine from the other  p r o t e i n s on p o l y a c r y l a m i d e  basic  g e l electrophoresis i n d i c a t e d that  a p r e p a r a t i v e method u t i l i z i n g sible.  B i o g e l P-10  (d) although producing  these reasons other methods were r e q u i r e d  The  and  t h i s s e p a r a t i o n might be  I d e n t i f i c a t i o n of the area of the g e l  fea-  containing  p r o t e i n s a f t e r e l e c t r o p h o r e s i s i s normally made by s t a i n i n g the g e l completely with Amido Black 10B,  an a n i o n i c dye  binds s t r o n g l y to c a t i o n i c groups i n p r o t e i n s but can e l u t e d from the g e l m a t r i x . the s u r f a c e of p o l y a c r y l a m i d e dye  which penetrated  I t was  which  be  p o s s i b l e to s t a i n only  g e l s w i t h Wool F a s t Blue BL,  the g e l extremely slowly and  which  a  was  e a s i l y washed from the s u r f a c e w i t h water a f t e r a b r i e f  stain-  ing period.  from  the  i n d i c a t e d area of the g e l by e l u t i o n of a mince of t h i s  gel  The  protamine c o u l d then be recovered  i n a s m a l l column. and  Subsequent d e t e r m i n a t i o n  of r a d i o a c t i v i t y  a r g i n i n e content e s t a b l i s h e d the s p e c i f i c a c t i v i t y of  l a b e l l i n g of protamine. g e l was  The  recovery  of protamine from  i n the order of 50 to 80 percent  and  adequate amount of r a d i o a c t i v e protamine from each This preparative  the  the c a p a c i t y  the g e l e l e c t r o p h o r e s i s system ensured the r e c o v e r y  g e l e l e c t r o p h o r e s i s method may  f u r t h e r a p p l i c a t i o n i n the p u r i f i c a t i o n of other  of  the  of  an  incubation.  w e l l have proteins.,  - 45 f o r i t does not r e q u i r e the e l a b o r a t e apparatus more l y used  frequent-  (133).  Preliminary testis cell  experiments i n which 1.0 ml a l i q u o t s of a  suspension  (1 ml packed c e l l s per 8 ml f i n a l  pension) from n a t u r a l l y maturing 0. nerka arginine-H  3  were incubated  (5 yC per i n c u b a t i o n , s p e c i f i c a c t i v i t y  mMole, f i n a l c o n c e n t r a t i o n  suswith  (1.4 C/  i n i n c u b a t i o n 3.5 x 10 ^ M), had  i n d i c a t e d t h a t the remarkably i n t e n s e i n c o r p o r a t i o n of a r g i n ine-H  3  i n t o protamine had e n t i r e l y d e p l e t e d  the c e l l  suspen-  s i o n of the added r a d i o a c t i v e i s o t o p e w i t h i n 30 minutes. t h i s reason i t was necessary t o d i l u t e the a r g i n i n e - H unlabelled arginine to give a f i n a l concentration c u b a t i o n medium of 1 x 10 ^ M.  Using  studied.  (chinook  i n the i n -  3  salmon) the time  i n t o protamine was  As shown i n F i g u r e 5 t h i s i n c o r p o r a t i o n  over a f o u r hour p e r i o d .  with  3  c e l l s from the t e s t e s  of n a t u r a l l y maturing 0. tshawytscha course of i n c o r p o r a t i o n of a r g i n i n e - H  For  continued  Thus suspensions o f i n t a c t sperma-  t i d c e l l s o f f e r e d a means of d e l i n e a t i n g some of the charact e r i s t i c s of protamine b i o s y n t h e s i s . (.c) E f f e c t of I n h i b i t o r s on Protamine The  Biosynthesis  b i o s y n t h e s i s of protamine i n the presence of i n h i -  b i t o r s o f p r o t e i n s y n t h e s i s has a l s o been examined with  sus-  pensions of these t e s t i s c e l l s from 0. tshawytscha.  In F i g -  ure  3  5, the time course of i n c o r p o r a t i o n of a r g i n i n e - H  protamine i n the presence of puromycin or actinomycin  into D, both  -5 at a c o n c e n t r a t i o n  of 5 x 10  M, i s shown.  P r e l i m i n a r y ex-  periments had i n d i c a t e d t h a t there was a time l a g o f about 30 minutes b e f o r e  the i n h i b i t o r y a c t i o n of puromycin on p r o -  4  -  46  -  CD C  Time , minutes  F i g u r e 5 - Time course of i n c o r p o r a t i o n of L - a r g i n i n e - H i n t o protamine cells.  by a suspension of 0.  Protamine  was  tshawytscha  testis  p u r i f i e d by the e l e c t r o p h o r e s i s i n  p o l y a c r y l a m i d e of the 0.2  M HC1  e x t r a c t s of each  t i o n , and the s p e c i f i c a c t i v i t y of the l a b e l l i n g by r a d i o a c t i v i t y  3  incubadetermined  and a r g i n i n e d e t e r m i n a t i o n s on each sample.  - 47 tamine s y n t h e s i s was seen. c r i b e d here, the c e l l  In a l l the experiments  t o be des-  suspensions were p r e i n c u b a t e d w i t h the  r e s p e c t i v e i n h i b i t o r s a t 0°C f o r 90 minutes p r i o r t o a d d i t i o n of  r a d i o a c t i v e p r e c u r s o r amino a c i d s , thus e n s u r i n g adequate  permeation  of a l l c e l l s with the i n h i b i t o r s .  There was a  strong i n h i b i t i o n of the i n c o r p o r a t i o n of a r g i n i n e - H protamine  3  by puromycin, but actinomycin D had no such  T h i s l a c k of i n h i b i t i o n of the s y n t h e s i s of protamine  into effect. i s not  due t o i m p e r m e a b i l i t y of the t e s t e s c e l l s t o a c t i n o m y c i n In  c o n t r o l experiments  D.  w i t h s i m i l a r c e l l s actinomycin D a t a -5  c o n c e n t r a t i o n of 5 x 10 uridine-H In  3  i n t o RNA  M, i n h i b i t e d the i n c o r p o r a t i o n of  77 percent  (147).  order t o demonstrate f u r t h e r the i n h i b i t i o n of p r o t a -  mine b i o s y n t h e s i s by i n h i b i t o r s of p r o t e i n s y n t h e s i s , the four i n h i b i t o r s , c y c l o h e x i m i d e , puromycin,  chloramphenicol,  and actinomycin D, were t e s t e d a t d i f f e r e n t c o n c e n t r a t i o n s u s i n g a suspension of t e s t i s c e l l s from S. g a i v d n e v i i  (steel-  head) which had been r e c e i v i n g i n j e c t i o n s of the p i t u i t a r y gonadotrophin  extract.  As shown i n F i g u r e 6 these i n c u b a t i o n s  were c a r r i e d out f o r 90 minutes a t 20°C, and the i n c o r p o r a t i o n of a r g i n i n e - H  i n t o protamine  i n the presence  of these  i n h i b i t o r s has been p l o t t e d as percent of the i n c o r p o r a t i o n in control incubations lacking Cycloheximide  inhibitor.  was by f a r the most potent i n h i b i t o r  pro-  ducing 50 percent i n h i b i t i o n a t a c o n c e n t r a t i o n of only 4 x -7 10  M.  Bennett  et al  (148) found t h a t i n mammalian c e l l s  a l s o , cycloheximide was more e f f e c t i v e than puromycin as an  - 48 -  —i  r  1  ocfanomyctn 0  O  ^100 chloramphenicol  O  e  o  40  Q.  8  20  puromycin  -7 -6 -5 L o g Concentration  -4 -3 -2 -I Inhibitor, Moles/liter  F i g u r e 6 - I n c o r p o r a t i o n of L - a r g i n i n e - H by S. g a i r d n e r i i concentrations  3  into  protamine  t e s t i s c e l l s i n the presence of v a r y i n g  of i n h i b i t o r s o f p r o t e i n s y n t h e s i s .  The  i n c u b a t i o n s were f o r 90 minutes under c o n d i t i o n s d e s c r i b e d in text.  - 49 i n h i b i t o r of p r o t e i n s y n t h e s i s .  However the i n h i b i t i o n i n  f i s h t e s t i s c e l l s occurs a t a c o n c e n t r a t i o n of two  cycloheximide  orders of magnitude below t h a t f o r the mammalian systems.  Chloramphenicol, s y n t h e s i s , was  the potent i n h i b i t o r of b a c t e r i a l  protein  o n l y e f f e c t i v e at c o n c e n t r a t i o n s g r e a t e r than  those f o r puromycin.  Actinomycin  D d i d not i n h i b i t  protamine  b i o s y n t h e s i s a t any of the c o n c e n t r a t i o n s t e s t e d , although i t had a marked e f f e c t on RNA suspensions The  as noted  s y n t h e s i s i n these same c e l l  above.  i n h i b i t i o n of protamine  b i o s y n t h e s i s by the three  i n h i b i t o r s of p r o t e i n s y n t h e s i s puromycin, c y c l o h e x i m i d e , chloramphenicol  and  can be taken as good evidence f o r the i n v o l v e -  ment of the messenger RNA, t h e s i s of protamine.  tRNA, ribosomal system i n the syn-  S i m i l a r c o n c l u s i o n s r e g a r d i n g the mech-  anism of s y n t h e s i s of i n s u l i n  (142)  and ACTH (143)  have been  made f o l l o w i n g the o b s e r v a t i o n t h a t the s y n t h e s i s of these small polypeptides i s s i m i l a r l y  i n h i b i t e d by puromycin.  Puromycin, cycloheximide and chloramphenicol a c t upon the t r a n s f e r of aminoacylgroups linkage  (149).  Puromycin c o n s i s t s of an  l i n k e d to an amino a c i d  appear to  from tRNA to p e p t i d e aminonucleoside  (150), and the c l o s e s t r u c t u r a l  emblance between puromycin and  res-  the amino a c i d b e a r i n g end of  tRNA l e d t o the o r i g i n a l p r o p o s a l t h a t t h i s a n t i b i o t i c a c t s by i n h i b i t i n g p r o t e i n s y n t h e s i s s p e c i f i c a l l y a t a s i t e i n v o l v i n g tRNA  (151) .  A c t i n g as an analogue, of aminoacy1-tRNA  puromycin forms a. "peptide" bond becoming the C-terminus of the nascent p e p t i d e c h a i n .  The  f a i l u r e of puromycin to b i n d  -  to ribosomes causes plex of incomplete mycin  50 -  the r e l e a s e from the mRNA-ribosome comp e p t i d e c h a i n s terminated by t h i s  puro-  (152-154). The  i n h i b i t i o n of p r o t e i n s y n t h e s i s by both c y c l o h e x i -  mide and chloramphenicol has been l o c a l i z e d t o the  transfer  r e a c t i o n s i n v o l v i n g aminoacyl-tRNA, ribosomes and mRNA which r e s u l t i n p e p t i d e bond f o r m a t i o n .  The exact mechanism by  which these i n h i b i t o r s a c t has not as y e t been e s t a b l i s h e d as c l e a r l y as t h a t f o r puromycin.  Cycloheximide  and the  r e l a t e d g l u t a r i m i d e s , s t r e p t o v i t o c i n A and a c e t o x y c y c l o h e x i mide have a s i m i l a r spectrum t e i n synthesis i n v i v o  of a c t i v i t y .  They i n h i b i t  i n y e a s t , f u n g i and a v a r i e t y of  mammalian c e l l s , as w e l l as i n a number o f c e l l - f r e e tions  pro-  (149), but not i n b a c t e r i a  (155).  In the r a t l i v e r  c e l l - f r e e system c y c l o h e x i m i d e decreases  the puromycin-  induced r e l e a s e of p e p t i d e s from ribosomes the breakdown o f ribosomal aggregates  prepara-  (156), p r e v e n t i n g  or polyribosomes  (157),  along w i t h a p a r a l l e l i n h i b i t i o n of amino a c i d i n c o r p o r a t i o n . These f i n d i n g s have been i n t e r p r e t e d as i n d i c a t i n g t h a t c y c l o heximide b l o c k s the "read-out" process by p r e v e n t i n g advancement of the ribosomes along the mRNA. r e f o r m a t i o n of polyribosomes  A t the same time the  i s also inhibited  (156).  In c o n t r a s t t o c y c l o h e x i m i d e , chloramphenicol  i s a very  potent i n h i b i t o r of b a c t e r i a l p r o t e i n s y n t h e s i s , w h i l e a t -4 s i m i l a r c o n c e n t r a t i o n s (10  -5 t o 10  most c e l l - f r e e systems from animals  M), i t has no e f f e c t on (158).  Although some  evidence would i n d i c a t e that, i t s mechanism of i n h i b i t i o n i s  - 51 dependent on i t s c a p a c i t y to compete w i t h the mRNA f o r ribosomal binding s i t e chloramphenicol  template  (159), i t i s more l i k e l y t h a t  a c t s a t a stage a f t e r b i n d i n g and d u r i n g pep-  t i d e bond s y n t h e s i s .  The  i n h i b i t i o n by chloramphenicol  the puromycin-induced  r e l e a s e of p e p t i d e s from ribosomes  as w e l l as the b i n d i n g of chloramphenicol .somal s u b u n i t  to the 50-S  of (160),  ribo-  (161,162) i n d i c a t e d t h a t t h e . i n h i b i t i o n i n v o l v e s  b i n d i n g of chloramphenicol  to a s p e c i f i c s i t e on the r i b o -  some, thus p r e v e n t i n g aminoacyl-tRNA b i n d i n g and  subsequent  p e p t i d e bond f o r m a t i o n . Miescher "nucleus" was  f i r s t conceived the i d e a t h a t a  protamine  a l r e a d y p r e s e n t i n the muscle p r o t e i n s of  t u r i n g salmon, and  ma-  t h a t by a d e g r a d a t i v e process accompany-  i n g the atrophy of these muscles p r i o r t o spawning such a protamine testes  nucleus c o u l d be r e l e a s e d and  (1,4).  Kossel  h i s t o n e molecules  t r a n s p o r t e d t o the  (2) assumed t h a t the more complex  p r e s e n t i n the immature t e s t e s a l s o  t a i n e d t h i s protamine  nucleus, thus r e p r e s e n t i n g as  mediate stage i n t h i s d e g r a d a t i v e p r o c e s s . t i o n of h i s t o n e i n t o protamine t o g e n e s i s would be merely the h i s t o n e molecules.  The  con-  inter-  transforma-  d u r i n g the course of sperma-  a removal of c e r t a i n p o r t i o n s of  T h i s concept has been a g a i n c o n s i d e r e d  i n a review of t h i s f i e l d  as r e c e n t l y as 1966  (5), but w i t h  the r a p i d advances of m o l e c u l a r b i o l o g y i n the l a s t decade and  i t s c o n t r i b u t i o n to the understanding  of a g e n e r a l  mechanism f o r p r o t e i n s y n t h e s i s , i t would be seen to have little  support.  The  evidence presented here,  certainly  - 52 i n d i c a t e s t h a t protamine sor  i s s y n t h e s i z e d de novo  from p r e c u r -  amino a c i d s and t h a t t h i s s y n t h e s i s proceeds  i n a manner  s i m i l a r to other p r o t e i n s u t i l i z i n g a mRNA, tRNA, r i b o s o m a l system. The f a c t t h a t a c t i n p m y c i n D d i d not i n h i b i t the s y n t h e s i s of  protamine  i s not l i k e l y t o be due  to the i m p e r m e a b i l i t y of  the c e l l s to t h i s i n h i b i t o r , f o r there was of  marked  inhibition  the i n c o r p o r a t i o n of r a d i o a c t i v e u r i d i n e i n t o RNA  trol  incubations.  The  testis cell  i n con-  suspension used i n these  s t u d i e s d i d c o n t a i n a somewhat heterogeneous  p o p u l a t i o n of  c e l l s a t a l l stages of m a t u r a t i o n although spermatids were c e r t a i n l y the major c e l l  type p r e s e n t .  The p o s s i b i l i t y r e -  mains t h a t the spermatid c e l l s s y n t h e s i z i n g protamine  could  be impermeable t o Actinomycin D w h i l e c e l l s a t e a r l i e r stages of m a t u r a t i o n , s t i l l the i n h i b i t i o n of RNA found t h a t t h e r e was at The  permeable to the i n h i b i t o r , show  synthesis. no RNA  Ando and Hashimoto  (25)  p r e s e n t i n the t e s t i s c e l l  a stage of maturation when protamine l a c k of i n h i b i t i o n of protamine  was  being s y n t h e s i z e d .  s y n t h e s i s by  actinomycin  D i s t h e r e f o r e c o n s i s t e n t w i t h these f i n d i n g s , f o r the c e l l n u c l e i must be i n a c t i v e i n RNA stage i n spermatogenesis.  synthesis at t h i s  A study of spermatogenesis  mouse t e s t e s a l s o i n d i c a t e d t h a t there was  nuclei  testis late in  a s y n t h e s i s of  a r g i n i n e - r i c h h i s t o n e s i n the absence of any new  RNA  synthesis  (163) . Since the s i t e of a c t i o n of a c t i n o m y c i n D as an of  inhibitor  p r o t e i n s y n t h e s i s has c l e a r l y been shown t o be upon the  -  53 -  t r a n s c r i p t i o n o f one strand of the DNA sequence i n t o RNA by DNA-dependent RNA polymerase  (164), t h i s lack of i n h i b i t i o n  c o u l d be i n t e r p r e t e d as i n d i c a t i n g t h a t a p u t a t i v e messenger RNA  f o r protamine, a l r e a d y  long l i f e  synthesized, might have a r e l a t i v e l y  i n the t e s t i s c e l l s .  Such s t a b l e mRNAs are known  i n mammalian r e t i c u l o c y t e s which s y n t h e s i z e absence of any RNA s y n t h e s i s  g l o b i n i n the  ( 1 6 5 ) , as w e l l as i n lens e p i -  thelial cells differentiating  to fibre cells  l a r i t y between f i b r o g e n e s i s i n the  (166) .  A simi-  l e n s , the m a t u r a t i o n of  r e t i c u l o c y t e s , and the m a t u r a t i o n of s k i n c e l l s has been noted (166) .  A l l these c e l l s have i n common the a b i l i t y  size t i s s u e - s p e c i f i c proteins and  such as c r y s t a l l i n s ,  k e r a t i n s , and the s y n t h e s i s  s t a b l e mRNA templates.  t o synthehemoglobin  of these p r o t e i n s occurs on  Furthermore, they a l l r e p r e s e n t  stages o f c e l l u l a r d i f f e r e n t i a t i o n and r e s u l t i n the of a c e l l which no longer  replicates.  final  formation  I n t h i s r e s p e c t the  marked t i s s u e s p e c i f i c i t y o f protamine and i t s s y n t h e s i s on a s t a b l e mRNA template might be analogous.  C e r t a i n l y the  d i f f e r e n t i a t i o n o f spermatogonia t o mature spermatozoa i s a f u r t h e r exapmple of a t e r m i n a l c e l l u l a r d i f f e r e n t i a t i o n sulting in a c e l l  type which no longer  replicates.  That  d i v i s i o n may p l a y a prominent r o l e i n d i f f e r e n t i a t i n g systems has a l s o been r e c o g n i z e d (167) .  by Stockdale  They noted t h a t DNA s y n t h e s i s  i n mammary gland  epithelium  cell  cell  and Topper  and/or c e l l  i s necessary f o r the  division initiation  of a d i f f e r e n t i a t i o n c h a r a c t e r i z e d by c a s e i n s y n t h e s i s . was  re-  suggested t h a t many of the cues which e l i c i t  the  It  process  - 54 of  d i f f e r e n t i a t i o n may only a c t on c e l l s a t the time o f t h e i r  proliferation. An i n h i b i t o r y e f f e c t of a c t i n o m y c i n D on protamine sis at  might be expected  i n experiments  utilizing testis  the exact stage of m a t u r a t i o n when protamine  synthecells  synthesis i s  i n i t i a t e d w i t h the s y n t h e s i s of a s u i t a b l e mRNA template. A l t e r n a t i v e l y i t i s p o s s i b l e t h a t such messenger RNA f o r protamine might be t r a n s c r i b e d a t a s t i l l m e i o s i s and subsequent of  spermiogenesis.  e a r l i e r stage i n the Such an accumulation  messenger RNA templates p r i o r t o t h e i r u t i l i z a t i o n i n p r o -  t e i n s y n t h e s i s may be a c h a r a c t e r i s t i c event i n c e l l u l a r ferentiation  (168).  The presence  in unfertilized  dif-  sea u r c h i n  eggs o f s t o r e d mRNA which was a c t i v e i n p r o t e i n s y n t h e s i s during f e r t i l i z a t i o n , been e s t a b l i s h e d f r o g embryos  cleavage and e a r l y b l a s t u l a stages has  (169), and experiments  (170)  with e a r l y d e v e l o p i n g  a l s o i n d i c a t e d t h a t although mRNA was  s y n t h e s i z e d d u r i n g the f i r s t  stages of embryonic development,  i t was t e m p o r a r i l y i n a c t i v e .  F u r t h e r examples o f • a "masked"  or  storage form o f messenger RNA have been r e p o r t e d i n a wide  v a r i e t y of systems  (171).  T h i s would i n d i c a t e t h a t the con-  t r o l of p r o t e i n s y n t h e s i s i n c e r t a i n cases i s a t the  transla-  t i o n a l l e v e l r a t h e r than a t the p o i n t of t r a n s c r i p t i o n RNA  template by RNA  into  polymerase.  (d) The P u r i f i c a t i o n o f Protamine The  f i r s t p r e p a r a t i o n of protamine  by Miescher (1,4)  i n v o l v e d e x t r a c t i o n of salmon sperm wi.th d i l u t e h y d r o c h l o r i c  - 55 a c i d , n e u t r a l i z a t i o n and p r e c i p i t a t i o n of protamine w i t h platinum  chloride.  Subsequently, a v a r i e t y o f p u r i f i c a t i o n  methods have been used  (4), most of which i n v o l v e s e v e r a l  c i p i t a t i o n steps and the formation or metal complexes.  In g e n e r a l ,  pre-  of s a l t s such as p i c r a t e s  these methods were found  s a t i s f a c t o r y when f r e s h l y c o l l e c t e d m i l t was the source of protamine f o r i t c o n t a i n s  only one type o f c e l l ,  spermatozoa.  When t e s t i s t i s s u e i s used, p a r t i c u l a r l y t h a t of f i s h undergoing s e x u a l m a t u r a t i o n as i n these s t u d i e s , there a r e a l s o present and  both sperm c e l l s a t e a r l i e r stages of development  somatic t i s s u e c e l l s c o n t a i n i n g  Furthermore, as p o i n t e d  h i s t o n e - l i k e basic  out by Bonner et al _ (66)  the a c i d ex-  t r a c t of whole c e l l s or even of c e l l n u c l e i may y i e l d p a r a t i o n s which i n c l u d e other  acid soluble proteins  the b a s i c p r o t e i n s of ribosomes.  proteins.  pre-  such as  The method of p r e p a r a t i o n  of protamine u t i l i z i n g column chromatography on g e l f i l t r a t i o n media developed i n these s t u d i e s was p a r t i c u l a r l y  suit-  a b l e t h e r e f o r e when the source of protamine was the t e s t i s t i s s u e of f i s h undergoing spermatogenesis, r a t h e r than the f r e s h l y c o l l e c t e d m i l t of spawning a d u l t The  protamine from S. g a i v d n e v i i  rainbow t r o u t ) as w e l l as some s p e c i e s  fish.  (both s t e e l h e a d and of the P a c i f i c  salmon  have been prepared by column chromatography on g e l f i l t r a t i o n media.  The 0.2 M h y d r o c h l o r i c a c i d e x t r a c t s of these  t i s s u e s were f i r s t chromatographed on Sephadex G-25. seen i n F i g u r e  7 the p r o t e i n s  testis As i s  i n the a c i d e x t r a c t of n a t u r a l l y  maturing rainbow t r o u t t e s t e s a r e p a r t i a l l y r e s o l v e d  i n t o two  - 56 -  CHROMATOGRAPHY OF TESTES ACID EXTRACT ON G-25  RECHROMATOGRAPHY OF G-25 HISTONE PEAK ON BIOGEL P-IO  RECHROMATOGRAPHY F G-25 PROTAMINE PEAK ON BIOGEL P- 10  Elution Volume, ml  Figure 7 - Gel f i l t r a t i o n from n a t u r a l l y The i n i t i a l  maturing S. g a i r d n e r i i  0.2 M HC1  Sephadex G-25  p u r i f i c a t i o n of protamine  e x t r a c t was  (rainbow  chromatographed  on  (upper f i g u r e ) , and the pooled h i s t o n e  and protamine peaks were each rechromatographed B i o g e l P-10  trout).  (middle and lower  figures).  on  - 57 peaks.  -  Rechromatography of each of these peaks, pooled  indicated, further  on B i o g e l  resolves  w e l l a f t e r the  P-10  u s i n g 0.2  these two  M a c e t i c a c i d as  fractions.  contaminating p o r t i o n  eluant  Protamine i s of the  as  eluted  h i s t o n e peak.  In f a c t the p o s i t i o n of i t s e l u t i o n i n d i c a t e d  that  it  was  being r e t a r d e d i n t h i s g e l f i l t r a t i o n to a much g r e a t e r  ex-  t e n t than i t s m o l e c u l a r weight range, 4000 to 5000 (4), would have i n d i c a t e d .  In p r e l i m i n a r y  experiments i t had  been found  t h a t the p o s i t i o n of e l u t i o n of protamine from a column of Biogel  P-10  was  dependent on  the  c o n c e n t r a t i o n of  acetic  a c i d i n the  eluting solvent.  Small columns (15 x 0.5  Biogel  had  the  P-10  e f f l u e n t s was  been used and  d e t e c t e d by  staining  f r a c t i o n with Amido B l a c k 10B Schwartz and  Zabin  (172)  i n two  second peak.  P-2  the  w i t h 0.1  of  each  paper.  e l u t i o n of  argi-  M acetic acid  M resulted  increasing i n the  rethe  enhance-  f i r s t peak w i t h a c o r r e s p o n d i n g r e d u c t i o n of  the  With protamine, e l u t i o n of the column w i t h  1.0  i n a s i n g l e peak c l o s e l y f o l l o w i n g  h i s t o n e peak, e l u t i o n w i t h 0.5 tamine peaks, one or 0.1  e a r l y and  one  M a c e t i c a c i d l e d to two well retarded, while  Obviously a r g i n i n e ,  and  elution  h i s t o n e peak.  s i m i l a r l y protamine w i t h i t s high  content, i n t e r a c t w i t h the p o l y a c r y l a m i d e i n  the  pro-  M a c e t i c a c i d gave the best s e p a r a t i o n , a  s i n g l e protamine peak e l u t i n g w e l l a f t e r the  arginine  on  of  column  aliquots  n i n h y d r i n p o s i t i v e peaks, and  M acetic acid resulted  with 0.2  ml  after spotting  a c e t i c a c i d c o n c e n t r a t i o n to 1.0 ment of the  0.05  reported that  nine from a column of B i o g e l sulted  protamine i n the  cm)  an  - 58 anomalous manner, perhaps e l e c t r o s t a t i c , and advantage  has  been taken of t h i s i n the p r e p a r a t i o n of protamine. The e l e c t r o s t a t i c i n t e r a c t i o n s of the c r o s s - l i n k e d  dex-  t r a n g e l s of the Sephadex type, due to the presence of a few t e r m i n a l c a r b o x y l i c groups, have been noted  (173, 174) and a  s i m i l a r u t i l i z a t i o n of these i n t e r a c t i o n s f o r the r e v e r s i b l e r e t e n t i o n of low m o l e c u l a r weight b a s i c p r o t e i n s such as t o x i n s of s c o r p i o n s , r i b o n u c l e a s e and lysozyme have been r e ported  (175).  Both these e l e c t r o s t a t i c or i o n exchange  effects,  as w e l l as a d s o r p t i o n e f f e c t s w i t h the Sephadex g e l s have been investigated recently  (176).  The e x t e n t of p u r i f i c a t i o n i n t h i s two step graphy was sis. of  chromato-  a l s o monitored by p o l y a c r y l a m i d e g e l e l e c t r o p h o r e -  Samples from the v a r i o u s stages i n a t y p i c a l  preparation  protamine, i n t h i s case from t e s t e s of the hormonally i n -  duced S. g a i r d n e r i i , are shown i n F i g u r e 8.  The h i s t o n e and  protamine both p r e s e n t i n the i n i t i a l a c i d e x t r a c t were seen to  be f a i r l y c l e a n l y separated by Sephadex G-25  w h i l e rechromatography on B i o g e l P-10  chromatography,  achieved complete  puri-  f i c a t i o n of protamine. (e)  Amino A c i d Composition of P u r i f i e d  Protamine  As a l r e a d y p o i n t e d out i n the i n t r o d u c t i o n , knowledge of the  primary s t r u c t u r e of protamine, or more p r o p e r l y of each  component of protamine i s necessary f o r the complete c h a r a c t e r i z a t i o n of the protamine s y n t h e s i s system i n these t e s t i s In  cells.  p a r t i c u l a r any d e t a i l e d study of the t r a n s l a t i o n a l p r o c e s s  -  59  ^.2  E o  l_  5  CSJ OO  I  3=  CU  cu  CZ  CD  O  o  TO  oo  5 rr O  cu  CD  E  cz o oo  o  CD Q;  —V CD  •—  0 1  o  TO  TO  £  < £  O  TO  TO , i_ _ CD ClO "TO* "QJ  —  H—'  o  TO -•—'  O  X  - Protamine  • Histones  1  + Figure 8 - Polyacrylamide  g e l e l e c t r o p h o r e s i s a t pH  samples from the v a r i o u s stages fication  (see F i g u r e 7)  hormonally induced  S.  4.5  i n the g e l f i l t r a t i o n  puri-  of protamine from the t e s t e s of gaivdnevii..  •  of  -  60 -  w i l l require.the determination  o f the sequence of nucleo-  t i d e s i n the p u t a t i v e mRNA f o r protamine as w e l l as p a r a l l e l s t u d i e s o f the p r o t e i n coded by t h a t p a r t i c u l a r mRNA  template.  The protamines from a number o f d i f f e r e n t s p e c i e s of f i s h used over the p e r i o d of these  i n v e s t i g a t i o n s were p u r i -  f i e d by the g e l f i l t r a t i o n procedure d e s c r i b e d i n the preceding  s e c t i o n , and the amino a c i d compositions  determined.  The amount of each amino a c i d i n micromoles i s  shown i n Table amino a c i d .  o f these were  I as w e l l as the number o f r e s i d u e s of each  T h i s l a t t e r assignment a r b i t r a r i l y assumed t h a t  the t o t a l number o f amino a c i d s i n each of these  various  protamines was 33. The three components of protamine from P a c i f i c h e r r i n g Clupea tively  pallasii,  Y l , Y I I , and Z have r e s p e c -  31, 30 and 31 amino a c i d r e s i d u e s  l i m i n a r y data o f Ando  (7), w h i l e  the p r e -  (19) on the protamine of S. g a i r d n e r i i  (rainbow t r o u t ) and Oncorhynchus  keta  (chum salmon) i n d i c a t e s  t h a t the c h a i n l e n g t h i s s l i g h t l y g r e a t e r ; 32 t o 34 f o r S. gairdnerii  protamine, and 34 f o r one component o f the 0.  keta  protamine (19). The protamines from a l l t h r e e s p e c i e s o f f i s h were q u i t e s i m i l a r i n amino a c i d composition; and  a high content  of arginine  s m a l l e r amounts of a r e s t r i c t e d range of n e u t r a l a c i d s ,  s e r i n e , p r o l i n e , g l y c i n e , v a l i n e , a l a n i n e and i s o l e u c i n e . The presence o f the l a t t e r two amino a c i d s , a l a n i n e and i s o l e u c i n e , as f r a c t i o n a l r e s i d u e s i n a l l three protamines cated t h a t e i t h e r s e v e r a l components were present  indi-  i n each  p r e p a r a t i o n of protamine, or t h a t these p r e p a r a t i o n s  were  Table I Amino A c i d A n a l y s e s of Protamine Amino Acid  from Salmonoid  Fish  S. gaivdnevii (Steelhead Trout)  0. tshawytscha (Chinook Salmon)  umoles  Residues  umoles  Residues  2.312  22.1  1.824  22.1  1.148  21.8  .369  3.5  .279  3.4  .191  3.6  PRO  .281  2.7  .217  2.6  .140  2.7  GLY  .230  2.2  .183.  2.2  .118  2.2  ALA  .053  0.5  .048  0.6  .030  0.6  VAL  .192  1.8  .142  1.7  .093  1.8  I LEU  .016  0.2  .031  0.4  .015  0.3  Total  3.453  33.0  2.724  33.0  1.735  33.0  ARG SER  +  ' The h y d r o l y s i s c a r r i e d out  of S. gaivdnevii  protamine  f o r 16, 36, 60 and 84 hours.  0. kisuteh (Coho Salmon) umoles  i n 6.07M HC1 '.  a t 105° C  f o r a 6% l o s s of s e r i n e  a n a l y s e s of. 16  was  No changes i n the r e c o v e r y o f  any amino a c i d i n the subsequent a n a l y s e s were noted except f o r A correction  Residues  has t h e r e f o r e  been  serine.  a p p l i e d to a l l  hour h y d r o l y s a t e s of the v a r i o u s protamines.  - 62 impure.  However both the s i n g l e band of p r o t e i n o b t a i n e d i n  the p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s of p u r i f i e d  protamine  (Figure 8 ) , and the absence of any other amino a c i d s p r e s e n t in  t r a c e amounts i n these amino a c i d analyses would r u l e out  the l a t t e r p o s s i b i l i t y .  The number of r e s i d u e s of s e r i n e  (3.5, 3.4 and 3.6) and of p r o l i n e  (2.7, 2.6 and 2.7) as non-  i n t e g r a l v a l u e s was a f u r t h e r i n d i c a t i o n of the h e t e r o g e n e i t y w i t h i n each of these protamines. observed  The p o s s i b l i t y t h a t t h i s  h e t e r o g e n e i t y might o r i g i n a t e by each  f i s h having i t s own p a r t i c u l a r protamine for  protamine  individual  has been r u l e d out,  p r e p a r a t i o n s from the t e s t e s of s i n g l e  both S. g a i r d n e r i i  (16,177) and Clupea  show s e v e r a l components of The behaviour  pallasii  fish,  (16), a l s o  protamine.  of protamine  (steelhead t r o u t ) , 0. tshawytscha  prepared from S. and 0. k i s u t c h  gairdnerii upon g e l  f i l t r a t i o n w i t h Sephadex G-25 was c o n s i s t e n t w i t h a m o l e c u l a r weight of l e s s than 5000, and hence a c h a i n l e n g t h of 30 t o 35 amino a c i d s .  T h e r e f o r e the presence  of a l a n i n e and i s o -  l e u c i n e as f r a c t i o n a l r e s i d u e s i n d i c a t e d c l e a r l y t h a t s e v e r a l components were p r e s e n t .  In s p i t e of t h i s i n d i c a t e d h e t e r o -  g e n e i t y o n l y s i n g l e N-terminal and C - t e r m i n a l amino a c i d s were found f o r the protamine  from S. g a i r d n e r i i .  The amino  a c i d analyses i n a s u b t r a c t i v e Edman d e t e r m i n a t i o n of the Nt e r m i n a l group, shown i n Table I I , i n d i c a t e d t h a t p r o l i n e was the only N-terminal amino a c i d p r e s e n t .  T h i s r e s u l t was  firmed by r e a c t i o n of the i n t a c t protamine aminonaphthalene-5-sulfonyl  chloride  with  con-  1-dimethyl-  (127) and the subsequent  -  63  -  Table II S u b t r a c t i v e Edman D e t e r m i n a t i o n of N-Terminal Amino A c i d s o f S. g a i v d n e v i i  (Steelhead Trout) Protamine  Amino A c i d  Residues I n t a c t Protamine  ARG  22.  1  A f t e r Edman 22.2  SER  3.5  3.4  PRO  2.7  1.7  GLY  2.2  2.1  ALA  0.5  0.5  VAL  1.8  1.8  ILEU  0.2  0.2  3 3 . 0  31.9  Total  demonstration of DNS-proline.  The  thin layer  separations  of the v a r i o u s marker DNS-amino a c i d s , as w e l l as the d e r i v a t i v e obtained ure  9.  DNS  from i n t a c t protamine, are shown i n F i g -  DNS-proline i s the o n l y major f l u o r e s c e n t spot  t a i n e d from protamine, and  i t i s c l e a r l y resolved  s o l v e n t system from the DNS mine amino a c i d s .  The  in either  d e r i v a t i v e s of a l l other  C-terminal  amino a c i d of t h i s  mine i s a r g i n i n e f o r when the carboxypeptidase A and of protamine were s u b j e c t e d  to e l e c t r o p h o r e s i s a t pH  (Figure 10),  the only amino a c i d r e l e a s e d  q u a n t i t y was  i d e n t i f i e d as  ob-  protaprotaB digest 6.5  in significant  arginine.  (f) Attempted F r a c t i o n a t i o n of Salmonoid Protamines The Clupea  f r a c t i o n a t i o n of protamine present  pallasii  ( P a c i f i c herring)  peine Y l , Y I I , and Clupeine  Z was  Z has  obtained  while clupeines  Y l and  i n the  i n t o three components, c l u -  been r e p o r t e d  by Ando et al  (7,18).  (6,7,18)  a f t e r chromatography on alumina YII were obtained  The  main h u r d l e  (6),  by chromatography  of the t r i n i t r o p h e n y l a t e d d e r i v a t i v e s of c l u p e i n e methylcellulose  t e s t e s of  to any  primary s t r u c t u r e of protamine thus overcome,  on  carboxy-  study of  the  Ando proceeded  to determine the amino a c i d sequences of each of these components of protamine.  As might be expected f o r any  with such a predominance of one i n sequence were not marked. sequences has  differences  A comparison of these  been shown e a r l i e r  components, YII and  amino a c i d , the  in Figure  1.  Z, have i d e n t i c a l primary  protein  Two  three of these  structures  Solvent B  Solvent A  Figure layer  9 - Separation chromatography.  v a t i v e s i s as f o l l o w s : (4) protamine,  (5)  of DNS-amino a c i d s by t h i n The order  of the DNS-deri-  (1) g l y , (2) a l a / (3)  pro,  , (6) a r g , (7) v a l , and  (8) s e r . The l i n e of spots c l o s e t o the o r i g i n i n the s o l v e n t system A i s due t o the s u l f o n i c acid  derivative,  w h i l e i n s o l v e n t system B , t h i s  same m a t e r i a l gives r i s e t o the l i n e of f a s t ^ migrating  spots.  - 66 -  CP  0«  #  A  Arg  *  0*  _  a s e  C P  a  s  e  B  0  F i g u r e 10 - Diagram of e l e c t r o p h o r e t o g r a m stained with ninhydrin a f t e r electrophoresis at pH 6.5, 20 min a t 62 v o l t s / c m . S. g a i r d n e r i i  The d i g e s t s o f  protamine w i t h carboxypeptidase A  and carboxypeptidase B were a p p l i e d t o the paper along w i t h the marker of a r g i n i n e .  A very  faint  band o f n i n h y d r i n p o s i t i v e m a t e r i a l i n the n e u t r a l cl S  r e g i o n was seen i n both the C P digests.  6  a o c  cl S  6  A and C P " ° " B  - 67 except f o r the s u b s t i t u t i o n of 5 N - t e r m i n a l r e s i d u e s i n the YII c h a i n by 6 i n the Z c h a i n , w h i l e the t h i r d component YI d i f f e r s more markedly from these two. Although the amino a c i d composition (Table I) had  indi-  cated t h a t protamine from each of these s p e c i e s of salmonoid f i s h was  heterogeneous, e l e c t r o p h o r e s i s of the p u r i f i e d pro-  tamines i n p o l y a c r y l a m i d e g e l s r e s u l t e d band of p r o t e i n  (Figure 8).  I t was  i n only a s i n g l e  probable t h e r e f o r e  the components of protamine of any one s p e c i e s of f i s h be of f a i r l y  s i m i l a r o v e r a l l composition.  that would  In an attempted  f r a c t i o n a t i o n of these components, protamine from the t e s t e s of 0. tshawytscha, chromatography  p u r i f i e d by the two-step g e l f i l t r a t i o n d e s c r i b e d i n (d) above, v/as chromatographed  on carboxymethyl Sephadex. out at a h i g h pH  The chromatography  was  carried  (11.4), approaching the i s o e l e c t r i c  point  of protamine, i n the hope t h a t s m a l l d i f f e r e n c e s i n the charge of each component might a l l o w a f r a c t i o n a t i o n of protamine. The e l u t i o n v/ith a g r a d i e n t of sodium c h l o r i d e d i d not r e s u l t however i n any f r a c t i o n a t i o n .  The protamine was  e l u t e d as  shown i n F i g u r e 11 i n a s i n g l e peak w i t h no evidence o f even any p a r t i a l f r a c t i o n a t i o n .  I t would appear t h a t i n t h i s  s p e c i e s a t any r a t e , the components of protamine have, a t the most, only very l i m i t e d d i f f e r e n c e s i n o v e r a l l charge.  Ando  has, i n c o n t r a s t , been a b l e to separate the t r i n i t r o p h e n y l a t e d components of protamine from Clupea  pallasii  s i m i l a r i o n exchange  (7,18).  chromatography  v e r y w e l l by  Chromatographic s e p a r a t i o n s of the components of p r o t a -  -  0  100  68 -  200  300  400  Elution Volume , ml F i g u r e 11 - Chromatography of protamine from 0. on carboxymethyl Sephadex. gradient  tshawytscha  The column was e l u t e d w i t h a  of NaCl i n 0.1 M N a C 0 2  3  b u f f e r a t pH 11.4.  Protein  c o n c e n t r a t i o n i n the e f f l u e n t f r a c t i o n s was estimated by a Sakaguchi r e a c t i o n  (125) .  -  mine from h e r r i n g  69  -  (14,16), salmon  (14), and t r o u t  (177)  columns of alumina e l u t e d w i t h potassium phosphate have been r e p o r t e d . t e s t e s was 0.45  i n F i g u r e 12.  tshawytscha  on a column of alumina u s i n g a  M d i p o t a s s i u m hydrogen phosphate as e l u a n t . as w e l l as rechromatography  (A) and decending  buffers  The p u r i f i e d protamine from 0.  chromatographed  chromatography  on  The  initial  of the ascending  (B) p o r t i o n s of t h i s s i n g l e peak are shown  I t appeared t h a t t h e r e had been some l i m i t e d  f r a c t i o n a t i o n of the protamine i n t h i s chromatographic for  the A and-B p o r t i o n s from the i n i t i a l  eluted at d i f f e r e n t T h i s work was  system  chromatography  were  positions. not pursued f u r t h e r when i t v/as l e a r n e d  t h a t s u c c e s s f u l f r a c t i o n a t i o n of protamine from both S. nerii  (rainbow t r o u t ) and 0. keta  (chum salmon) had been  achieved by Ando and h i s a s s o c i a t e s the S.  (19).  A comparison of  amino a c i d compositions of the p u r i f i e d protamines gaivdnevii  (steelhead  trout),  0.  tshawytscha  and  from  0.  kisuteh  with those o b t a i n e d f o r the separated components of S. nevii  (rainbow t r o u t ) and 0. keta  gaivd-  gaivd-  are shown i n T a b l e I I I .  The o v e r a l l composition of the protamines prepared i n these s t u d i e s was the  v e r y s i m i l a r t o those g i v e n by Ando f o r each of  components of t r o u t and salmon protamine.  The two amino  acids, p r e s e n t as f r a c t i o n a l r e s i d u e s i n S, g a i v d n e v i i  (steel-  head t r o u t ) protamine, i s o l e u c i n e and a l a n i n e , occur as s i n g l e r e s i d u e s i n component I and component II r e s p e c t i v e l y of  the S. g a i v d n e v i i  (rainbow t r o u t )  protamine.  Steelhead t r o u t and rainbow t r o u t belong to the same  - 70 -  Rechromatography B  Elution Volume , ml F i g u r e 12 - Chromatography of 0.  tshawytscha  protamine on alumina e l u t i n g w i t h 0.45 M K H P O i * 2  Table I I I Amino A c i d Composition of Protamines from Salmonoid Amino Acid ARG  S. g a i r d n e r i i (Steelhead Trout) 22.1 Res.  S.  gairdnerii  (Rainbow Trout) II 22-23 Res.  21-22 Res,  0.  tshawytscha  (Chinook Salmon)  22.1 Res.  Fish 0.  kisutch  (Coho Salmon)  21.8 Res.  + 0.  keta  (Chum Salmon) 23 Res.  SER  3.5  4  4  3.4  3.6  4  PRO  2.7  3  2  2.6  2.7  3  GLY  2.2  2  2  2.2  2.2  2  ALA  0.5  1  0.6  0.6  VAL  1.8  1  2  1.7  1.8  ILEU  0.2  1  0.4  0.3  33.0  33.0  Total  33.0  from r e f e r e n c e (19)  33-34  32-33  i  34  - 72 species, and  Salmo  gairdnerii  Europe), but  -  ( a l s o c a l l e d Salmo  show behaviour d i f f e r e n c e s ,  anadromous, spending a p r o p o r t i o n the  latter  s o l e l y a f r e s h water v a r i e t y .  f o r these two  fish  (178), and  of protamine i n t h i s work, and  i n Ando's work was  not  commonly regarded  (179)  the genus Salmo, 0.  kisutch  and  n e r i i .protamine  and 0.  keta  unexpected.  The  i n Japan  former being  i n s a l t water,  Starch  gel electro-  showed i d e n t i c a l pat-  therefore  i n amino a c i d compositions between the paration  the  of i t s l i f e  phoretograms of muscle myogen p r o t e i n s terns  irideus  the  steelhead  similarity trout pre-  t h a t f o r rainbow t r o u t genus Oncorhynchus  to have a r i s e n from the a n c e s t o r s of  the  s i m i l a r i t y between 0.  protamine on  the  one  tshawytscha,  hand and  S.  on the other i s f u r t h e r evidence of t h i s  close relationship.  is  gaird-  - 73 PART I I PHOSPHORYLATION OF PROTAMINE I n t e r e s t i n the p h o s p h o r y l a t i o n first (78)  of n u c l e a r p r o t e i n s was  s t i m u l a t e d by the r e p o r t i n 1965 of Huang and Bonner t h a t n a t i v e h i s t o n e molecules as they occur  i n the nu-  c l e o h i s t o n e component of pea bud chromatin c o n t a i n RNA molecules c h e m i c a l l y  l i n k e d t o them.  The p o s s i b i l i t y o f  i n v e s t i g a t i n g whether such a s i m i l a r protamine-RNA complex might e x i s t i n the t e s t i s c e l l s of maturing s t e e l h e a d  trout  seemed a t t r a c t i v e , f o r the c e l l  uniquely  suspension system was  s u i t a b l e f o r a study of the i n c o r p o r a t i o n of p r e c u r s o r ganic phosphate-P  32  i n t o a protamine complex.  of K l e i n s m i t h , A l l f r e y and Mirsky  inor-  The experiments  (75) and S t e v e l y and Stocken  (76) had a l s o c l e a r l y demonstrated the i n c o r p o r a t i o n of phosphate-P The  3 2  i n t o t h e h i s t o n e s o f c a l f and r a t thymus n u c l e i .  histone-bound RNA of the former study  (78) was o n l y ob-  t a i n e d when the customary a c i d e x t r a c t i o n of chromatin was abandoned i n favour  of a method employing high  ionic  strength  s o l u t i o n s of cesium c h l o r i d e t o d i s s o c i a t e h i s t o n e from the DNA i n chromatin, the histone-RNA l i n k a g e being acid conditions.  In c o n t r a s t t o t h i s , the s i t e of l i n k a g e of  phosphate i n the l a t t e r two s t u d i e s o-phOsphoserine  unstable i n  (75,76) was shown t o be  (75,76) and o-phosphothreonine  (75),  covalent  l i n k a g e s which a r e s t a b l e i n the a c i d c o n d i t i o n s used f o r e x t r a c t i o n of b a s i c p r o t e i n s .  The presence of o-phospho-  s e r i n e i n the p a r t i a l a c i d h y d r o l y s a t e s a v a i l a b l e p r e p a r a t i o n of protamine  of a commercially  (salmine) had a l s o been  - 74 noted by Murray MATERIALS  (180) .  and METHODS  (a) I n c u b a t i o n  Procedures  C e l l s a t a stage a c t i v e i n the s y n t h e s i s of protamine were obtained  from the t e s t e s of S. g a i v d n e v i i  (steelhead  t r o u t ) i n which spermatogenesis had been induced. had  F i s h which  r e c e i v e d i n j e c t i o n s of the salmon p i t u i t a r y e x t r a c t f o r  7 t o 10 weeks, were k i l l e d ,  the t e s t e s e x c i s e d and suspen-  s i o n s of i n t a c t c e l l s made i n the manner a l r e a d y (Part I ) .  As phosphate was present  i n the Hanks medium used  i n those e a r l i e r s t u d i e s , a m o d i f i e d the i n c u b a t i o n s  described  b u f f e r was adopted f o r  c o n t a i n i n g 0.14 M sodium c h l o r i d e , 5.4 mM  potassium c h l o r i d e , 0.81 mM magnesium s u l p h a t e , cium c h l o r i d e , 5.6 mM g l u c o s e  and 4.2 mM sodium  1.26 mM  cal-  bicarbonate  (pH 7.0 t o 7.2). (i) Large s c a l e The  incubations:  c e l l suspension,  centrifuged  (900 g, 5 min) and r e -  suspended i n the same medium was a t a f i n a l d i l u t i o n of 8 ml packed c e l l s  i n 35 ml f i n a l  c a r r i e d out i n stoppered  suspension.  The i n c u b a t i o n s were  50 ml Erylenmeyer f l a s k s on a New  Brunswick water bath, Model G77, w i t h g y r a t o r y 20°C. P  3 2  L a b e l l e d compounds, 40 t o 200 yC i n o r g a n i c phosphate-  (Atomic Energy of Canada, Ltd., c a r r i e r f r e e ) or 10 uC  L-serine-C at  shaking a t  1  ** (New England Nuclear,  the s t a r t of the i n c u b a t i o n s .  a f t e r 1 t o 3 hours by c h i l l i n g  131.1 mC/mMole) were added The i n c u b a t i o n s v/ere stopped  t o 0°C on i c e , twice  centri-  - 75 fuging  (500  g,  5 min)  and  -  resuspending i n the  incubation  -2 medium made 10  M i n either non-radioactive  s e r i n e as a p p r o p r i a t e .  The  phosphate or  c e l l s were then c o l l e c t e d by  c e n t r i f u g a t i o n f o r e x t r a c t i o n of the n u c l e a r (ii) The  I n d i v i d u a l small cell  incubations:  suspension from s t e e l h e a d  l a r l y prepared, was i n 4 ml  scale  trout testes,  simi-  used a t a d i l u t i o n of 1 ml packed  cells  f i n a l suspension.  pension were p i p e t t e d incubated a t 0°C  proteins.  Aliquots,  i n t o 13 x 100  f o r 90 minutes.  0.5 mm  ml,  of t h i s sus-  t e s t tubes and  Puromycin  N u t r i t i o n a l B i o c h e m i c a l s , Corp.) and  (dihydrochloride,  cycloheximide  t i o n a l B i o c h e m i c a l s , Corp.) were p r e s e n t a t a 4 5 of 10  M and  incubations.  5 x 10  period,  t i o n s were begun by the a d d i t i o n of 10 yC P ,  and  3 2  t r a n s f e r to the  were stopped a t v a r i o u s 1.0  M hydrochloric  (b) E x t r a c t i o n and  0.2  a c i d and  chilling  to  M hydrochloric  The  phosphate-  incubations  30 min).  ml  Proteins  of phospho-protamine: incubation  were  extracted  a c i d by homogenization i n 50  w i t h a T r i - R homogenizer) and  homogenate (15,000 g,  incuba-  0°C.  P u r i f i c a t i o n of Nuclear  P o t t e r - E l v e h j e m tubes with a g l a s s - T e f l o n 2 min  the  these  times by the a d d i t i o n of 0.2  washed c e l l p e l l e t s from the  w i t h 20 ml  concentration  inorganic  20°C water bath.  (i) Large s c a l e p r e p a r a t i o n The  (Nutri-  M r e s p e c t i v e l y i n c e r t a i n of  A f t e r the p r e i n c u b a t i o n  pre-  pestle  (5000  c e n t r i f u g a t i o n of  After a similar  ml  rpm, the  reextraction  of the p e l l e t s the combined supernatants were l y o p h i l i z e d .  -  76 -  The d r y r e s i d u e s were d i s s o l v e d i n water, c l a r i f i e d by a -brief c e n t r i f u g a t i o n  (500 g, 5 min), d e s a l t e d on a column  (40 x 2 cm) of Sephadex G-10 (Pharmacia) e l u t i n g w i t h water, and passed through e i t h e r a column  (10 x 2 cm) of Dowex 1 x 8  (Bio-Rad L a b o r a t o r i e s ) i n the a c e t a t e form, e l u t i n g w i t h water, or adsorbed on a column of carboxymethyl Sephadex (Pharmacia) i n the H  form, e l u t i n g f i r s t w i t h water and then  0.1 M h y d r o c h l o r i c a c i d . f r e e phosphate-P  The r e s u l t i n g p r o t e i n peak w i t h  o r s e r i n e - C * thus removed was l y o p h i l i z e d .  3 2  11  The b a s i c p r o t e i n s were then p u r i f i e d by the s e q u e n t i a l g e l f i l t r a t i o n on columns  of Sephadex G-2 5 (50 x 2.5 cm) i n  0.1 M a c e t i c a c i d and B i o g e l P-10 (30 x 2 cm) i n 0.2 M a c e t i c acid.  P r o t e i n c o n c e n t r a t i o n i n the e f f l u e n t f r a c t i o n s v/as  estimated by the absorbance a t 220 my i n a Beckman DB s p e c t r o photometer  a f t e r 10 f o l d d i l u t i o n of an a l i q u o t o f each f r a c -  t i o n w i t h water. or C  1k  S u i t a b l e a l i q u o t s f o r d e t e r m i n a t i o n of P  3 2  r a d i o a c t i v i t y were removed from each f r a c t i o n and  were p l a c e d i n g l a s s c o u n t i n g v i a l s . lation fluid  Ten ml Bray's  scintil-  (12 4) were added, and the samples counted i n a  Nuclear Chicago U n i l u x c o u n t e r . ( i i ) P r e p a r a t i o n of phospho-protamine  from i n d i v i d u a l  incubations: The i n c u b a t i o n s of t e s t i s c e l l i c phosphate-P  32  suspensions w i t h i n o r g a n -  had been stopped by the a d d i t i o n of 1.0 M  hydrochloric acid.  The a c i d e x t r a c t s of these c e l l  s i o n s were made by v i g o r o u s l y resuspending each  suspen-  individual  -  77  i n c u b a t i o n u s i n g a V o r t e x mixer gation  -  (Fisher).  After  centrifu-  (500 g, 5 min) and s i m i l a r r e e x t r a c t i o n of the p e l l e t s  w i t h 0.5 ml 0.5 M h y d r o c h l o r i c a c i d , the combined supernatants were l y o p h i l i z e d .  The p u r i f i c a t i o n of the protamine was then  performed as d e s c r i b e d i n P a r t I of t h i s t h e s i s , u s i n g t r o p h o r e s i s on p o l y a c r y l a m i d e g e l s changed  elec-  (the b u f f e r i n the g e l was  t o 0.05 M sodium a c e t a t e , pH 6.5) and r e c o v e r y o f  the protamine from the g e l by e l u t i o n of a mince of the a p p r o p r i a t e area of the g e l .  The subsequent d e t e r m i n a t i o n of  r a d i o a c t i v i t y , by c o u n t i n g i n Bray's s c i n t i l l a t i o n f l u i d  (124),  and of a r g i n i n e , by the Sakaguchi r e a c t i o n used p r e v i o u s l y , e s t a b l i s h e d the s p e c i f i c a c t i v i t y of the l a b e l l i n g of p r o t a mine w i t h  phosphate-P . 32  (c) P u r i f i c a t i o n of Phospho-peptides from Protamine The protamine, l a b e l l e d w i t h e i t h e r p h o s p h a t e - P serine-C  l l f  32  or  and which had been p u r i f i e d by the s e q u e n t i a l g e l  f i l t r a t i o n on Sephadex G-25 and B i o g e l P-10 was d i g e s t e d u s i n g a 1:50 r a t i o of t r y p s i n  (Worthington, 2x c r y s t a l l i z e d ,  s a l t f r e e ) t o p r o t e i n i n 0.1 M ammonium bicarbonate, pH 8.0, a t 37°C f o r 4 t o 10 hours.  The r e s u l t i n g t r y p t i c  phospho-  p e p t i d e s from protamine were p u r i f i e d by two methods: (i) P u r i f i c a t i o n by e l e c t r o p h o r e s i s and chromatography on paper: The protamine d i g e s t s were s p o t t e d i n a narrow from 10 t o 40 cm long on Whatman 3 MM paper. w e t t i n g the paper w i t h b u f f e r  After  strip carefully  ( p y r i d i n e / a c e t i c acid/water,  - 78 100:'4:'900, pH  6.5),  c o o l e d apparatus  high v o l t a g e e l e c t r o p h o r e s i s i n a V a r s o l -  a t pH  20 t o 60 minutes.  6.5  was  c a r r i e d out a t 62 v o l t s / c m f o r  Autoradiography  of the d r i e d e l e c t r o p h o r e -  tograms r e v e a l e d the l o c a t i o n of the major r a d i o a c t i v e pept i d e s as two bands only p a r t i a l l y r e s o l v e d i n the n e u t r a l region.  The  s t r i p of paper c o n t a i n i n g these p e p t i d e s  then c u t out and  s t i t c h e d t o a second  paper f o r descending g r a p h i c system  chromatography.  was  sheet of Whatman 3 The  initial  (n-butanol/formic acid/water,  chromato-  15:3:2) r e s u l t e d  i n two bands of r a d i o a c t i v e p e p t i d e s as r e v e a l e d by the radiography.  One  of these was  c u t out and  other, l o c a t e d a t the o r i g i n , was to another  sheet of paper.  a g a i n u s i n g a second 3:1:1).  system  MM  auto-  s e t a s i d e ; the  a l s o cut out and  Chromatography was  stitched  carried  out  ( n - b u t a n o l / a c e t i c acid/water,  T h i s l a t t e r system r e s u l t e d i n three p a r t i a l l y r e -  s o l v e d bands of r a d i o a c t i v e p e p t i d e s . t i o n s obtained i n t h i s descending e l u t e d from the paper w i t h 0.1 of Markham (181)  Each of the f o u r f r a c -  chromatography, was  M a c e t i c a c i d by the method  and r e r u n on h i g h v o l t a g e paper  p h o r e s i s a t pH 1.9  electro-  ( a c e t i c a c i d / f o r m i c acid/water,  75 v o l t s / c m , 30 min)  now  75:25:900,  which e f f e c t e d f u r t h e r r e s o l u t i o n .  The  major p e p t i d e s were again e l u t e d from the paper and s t o r e d at -20°C u n t i l f u r t h e r c h a r a c t e r i z e d . procedure  This p u r i f i c a t i o n  i s shown d i a g r a m a t i c a l l y i n F i g u r e  Kodak Royal Blue m e d i c a l X-ray  f i l m was  radiography of paper electrophoretograms, polyacrylamide g e l s .  19. used i n auto-  chromatograms or  The papers were s t a p l e d d i r e c t l y  to  -  79 -  the X-ray f i l m , the s t a p l e h o l e s s e r v i n g i n the subsequent •al-ignment o f the paper and f i l m ; the p o l y a c r y l a m i d e g e l was covered w i t h Saran wrap, a marker  of i n k made r a d i o a c t i v e w i t h  C -amino a c i d s a i d i n g i n i t s subsequent alignment. ll,  The times  of exposure v a r i e d from 12 hours t o 30 days depending on the amount of r a d i o a c t i v i t y p r e s e n t ; a t o t a l of approximately 1 0  6  counts over the d u r a t i o n of the exposure normally served t o g i v e a r e a s o n a b l y i n t e n s e spot of about 2.0 cm diameter on the X-ray  film.  ( i i ) P u r i f i c a t i o n by i o n exchange  chromatography:  The t r y p t i c d i g e s t from another p r e p a r a t i o n of phosphateP  3 2  l a b e l l e d protamine was chromatographed  on Dowex 50 x 2  (Bio-Rad L a b o r a t o r i e s ) a c c o r d i n g t o the method d e s c r i b e d by Schroeder et al ( 1 8 2 ) .  The j a c k e t e d column  (100 x 0.9 cm)  was maintained a t 37°C and e l u t e d w i t h a g r a d i e n t produced by a r r a n g i n g three 50 0 ml beakers connected i n s e r i e s and c o n t a i n i n g 300 ml of b u f f e r i n each as f o l l o w s :  v e s s e l 1-  p y r i d i n i u m a c e t a t e pH 3.1, 0.2 M; v e s s e l s 2 and 3 - p y r i d i n ium a c e t a t e pH 5.0, 2.0 M.  A f t e r completion of the g r a d i e n t ,  e l u t i o n of the column was c o n t i n u e d w i t h a f u r t h e r 2 50 ml of the pH 5.0 b u f f e r .  An a l i q u o t , 0.2 ml, of each e f f l u e n t  f r a c t i o n was removed f o r d e t e r m i n a t i o n of r a d i o a c t i v e phate-P before.  3 2  c o u n t i n g i n Bray's s c i n t i l l a t i o n f l u i d  phos-  (124) as  The a p p r o p r i a t e p o r t i o n s o f the e f f l u e n t were then  pooled, l y o p h i l i z e d , and the phospho-peptides r e d i s s o l v e d i n water and s t o r e d a t -20°C.  - 80 (d) I d e n t i f i c a t i o n and C h a r a c t e r i z a t i o n of P u r i f i e d Phosphopeptides The amino a c i d a n a l y s i s , phosphate d e t e r m i n a t i o n and Nt e r m i n a l amino a c i d i d e n t i f i c a t i o n c h a r a c t e r i z e d each of the p u r i f i e d phospho-peptides from e i t h e r paper or column chromatographic preparations. h y d r o l y z e d i n vacuo f o r 16 hours.  Samples f o r amino a c i d a n a l y s i s were  w i t h 6.0 M h y d r o c h l o r i c a c i d a t 105°C  The a n a l y s e s were c a r r i e d out e i t h e r on a  Technicon Auto A n a l y z e r , or a Beckman 120C Amino A c i d Analyzer.  The N - t e r m i n a l amino a c i d s of the phospho-peptides  were i d e n t i f i e d by r e a c t i o n w i t h 1-dimethylaminonaphthalene5 - s u l f o n y l c h l o r i d e a c c o r d i n g t o Gray and H a r t l e y  (127) w i t h  h y d r o l y s i s and subsequent i d e n t i f i c a t i o n of the DNS-amino a c i d s by t h i n l a y e r chromatography  (128).  Alternatively  end  groups were determined by a s u b t r a c t i v e Edman technique (129), as d e s c r i b e d i n P a r t I when the N - t e r m i n a l amino a c i d of i n t a c t protamine was  determined.  The phosphate c o n t e n t s of  both p u r i f i e d p r e p a r a t i o n s of protamine and the p u r i f i e d t r y p t i c p e p t i d e s were determined by e i t h e r the method of Gomori (18 3) ( a f t e r treatment of the protamine w i t h 1.0 sodium hydroxide, 100°C, 15 min, and n e u t r a l i z a t i o n w i t h  M 1.0  M h y d r o c h l o r i c a c i d ) or a l t e r n a t i v e l y by the method of Ames (184). (e) D e p h o s p h o r y l a t i o n of Protamine and  Phospho-peptides  The s t a b i l i t y of the c o v a l e n t phospho-protamine was  i n v e s t i g a t e d by h e a t i n g a l i q u o t s of the p u r i f i e d  mine-?  32  linkage prota-  i n 0.1 M h y d r o c h l o r i c a c i d and 0.1 M sodium hydroxide  - 81 f o r 15 minutes a t 100°C.  A f t e r n e u t r a l i z a t i o n w i t h sodium  hydroxide or h y d r o c h l o r i c a c i d as a p p r o p r i a t e , the samples were a p p l i e d t o Whatman 3 MM paper f o r h i g h v o l t a g e e l e c t r o p h o r e s i s a t pH 6.5 as d e s c r i b e d i n (c) above.  Autoradiography  of the d r i e d electrophoretogram r e v e a l e d the l o c a t i o n of the r a d i o a c t i v e p h o s p h a t e - P , w h i l e s t a i n i n g the paper w i t h 32  Amido Black 10B l o c a t e d the protamine. Protamine c o n t a i n i n g s e r i n e - C * was d i g e s t e d w i t h  tryp-  11  s i n as d e s c r i b e d i n (c) above, and t r e a t e d w i t h soybean trypsin inhibitor  (Worthington,  per mole of t r y p s i n ) .  3x c r y s t a l l i z e d ,  2 moles  The d i g e s t was then incubated f o r 60  minutes a t 45°C w i t h a 0.05 ml a l i q u o t of a 10 mg per ml suspension of E. c o l i  a l k a l i n e phosphatase  These dephosphorylated  (Worthington,  t r y p t i c p e p t i d e s from protamine  BAPC). v/ere  then examined w i t h h i g h v o l t a g e e l e c t r o p h o r e s i s on Whatman 3 MM paper a t pH 6.5 as s i m i l a r l y d e s c r i b e d f o r the phosphateP  3 2  l a b e l l e d protamine  digests.  RESULTS and DISCUSSION (a) P h o s p h o r y l a t i o n of H i s t o n e and Protamine Using suspensions of c e l l s from the maturing  t e s t e s of  s t e e l h e a d t r o u t which had been r e c e i v i n g the i n j e c t i o n s of p i t u i t a r y e x t r a c t , and which were a t a stage a c t i v e i n the b i o s y n t h e s i s of protamine,  i n c u b a t i o n s w i t h i n o r g a n i c phos-  phate-P  The i n c u b a t i o n c o n d i t i o n s were  32  were performed.  s i m i l a r to those a l r e a d y d e s c r i b e d f o r the i n c o r p o r a t i o n of l a b e l l e d p r e c u r s o r amino a c i d s i n t o protamine,  except t h a t  - 82 the i n c u b a t i o n medium was  modified,  - b u f f e r as t h i s would o b v i o u s l y phosphate. 0.2  The  and  by  a procedure a l r e a d y  and  t i o n and  utilized  was  32  in-  protamine f r a c t i o n s which The  y i e l d s a pure p r e p a r a t i o n  of  i t s c h a r a c t e r i s t i c amino a c i d composi-  the s i n g l e N - t e r m i n a l amino a c i d , p r o l i n e .  p u r i f i c a t i o n procedure has P a r t I,  on  t h i s two-stage chromatography.  rechromatography on B i o g e l P-10 protamine as judged by  by g e l f i l t r a t i o n  13 i n o r g a n i c p h o s p h a t e - P  i n t o both h i s t o n e  were w e l l separated  with  I.  i s seen i n F i g u r e  corporated  d i l u t e the added r a d i o a c t i v e  achieved  B i o g e l P-10,  i n the s t u d i e s of P a r t As  the phosphate  p u r i f i c a t i o n of the p r o t e i n s e x t r a c t e d  M h y d r o c h l o r i c a c i d was  Sephadex G-25  omitting  (c) of t h i s t h e s i s .  been more f u l l y Bonner et al  This  discussed (66)  in  have s t r e s s e d  the importance of u s i n g p u r i f i e d chromatin as a s t a r t i n g material for histone preparation.  The  a c i d e x t r a c t i o n of  whole t i s s u e , or even of n u c l e i , y i e l d s p r e p a r a t i o n s clude other  which i n -  a c i d - s o l u b l e p r o t e i n s such as the b a s i c  of ribosomes.  The  preparation  described  here was  a simple  a c i d e x t r a c t of the c e l l suspension i n c u b a t i o n and mediate step to p u r i f y chromatin was  proteins  an  not i n c l u d e d .  inter-  For  this  reason i t i s p o s s i b l e t h a t a p o r t i o n of the p r o t e i n e l u t e d i n the f i r s t peak form the Sephadex G-25 with phosphate-P  32  column and l a b e l l e d  might be non-histone p r o t e i n .  However  only those p r o t e i n s s o l u b l e i n water a f t e r l y o p h i l i z a t i o n of the a c i d e x t r a c t were used i n the  subsequent g e l  and most of the non-histone p r o t e i n s present  filtration  i n the i n i t i a l  RECHROMATOGRAPHY ON BIOGEL  ACID EXTRACTS ON G-25  25  P-IO  50  ELUTION V O L U M E . ML  Figure  13 - G e l f i l t r a t i o n chromatography P  3 2  - l a b e l l e d p r o t e i n s e x t r a c t e d from the  t e s t i s c e l l s of S. g a i v d n e v i i a f t e r i n c u b a t i o n w i t h i n o r g a n i c •  • A 2 2 0 ; o — — o CPM P . 3 2  Following  phosphate-P ,  i n i t i a l chromatography on Sephadex  32  G-25  the  f r a c t i o n s c o n t a i n i n g protamine were combined as i n d i c a t e d , l y o p h i l i z e d and rechromatographed on B i o g e l  P-10.  - 84 e x t r a c t , but i n s o l u b l e i n water, would be d i s c a r d e d a t t h i s point. phate-P  I t i s l i k e l y therefore that t h i s f i r s t 3 2  peak of phos-  r a d i o a c t i v i t y does i n f a c t r e p r e s e n t phosphory-  l a t i o n of h i s t o n e s . It  should be noted  t h a t the method of e x t r a c t i o n .with  a c i d used i n these s t u d i e s would not permit recovery of b a s i c protein-RNA complex as found by Huang and Bonner  the (78).  In view of the e x t e n s i v e p h o s p h o r y l a t i o n of protamine shown in  the f o l l o w i n g s e c t i o n s , the p h o s p h a t e - P  observed  here was  32  incorporation  b e l i e v e d to be a d i f f e r e n t phenomenon than  t h a t found by Huang and Bonner.  I t would be of  interest  n e v e r t h e l e s s to i s o l a t e these n u c l e a r p r o t e i n s from  testis  t i s s u e u s i n g the c o n d i t i o n s of h i g h i o n i c s t r e n g t h i n order to  preserve The  such a protein-RNA complex.  h i s t o n e s c o n s t i t u t e a heterogeneous group of p r o t e i n s  (5,66,72) although s t u d i e s has  some of the h e t e r o g e n e i t y  s i n c e been a t t r i b u t e d to contamination  somal p r o t e i n s , to a g g r e g a t i o n and  found  of h i s t o n e s w i t h one  to p r o t e o l y s i s of h i s t o n e s d u r i n g p r e p a r a t i o n  Nevertheless  by  ribo-  another, (66).  they do remain a somewhat complex group of b a s i c  p r o t e i n s , the f u l l attained.  i n early  c h a r a c t e r i z a t i o n of which has not y e t been  In c o n t r a s t the protamines are a r a t h e r  group of c l o s e l y r e l a t e d p r o t e i n s  (4,5)  and  the  simple  chemical  c h a r a c t e r i z a t i o n of these has proceeded much f u r t h e r (8). For t h i s reason a t t e n t i o n has been focussed only upon the p h o s p h o r y l a t i o n of protamine o c c u r i n g i n these t e s t i s preparations.  cell  - 85 (b) Nature of the Phospho-protamine Linkage Although the p o l y b a s i c nature of protamine might be expected t o l e a d t o i o n i c or e l e c t r o s t a t i c b i n d i n g o f i n o r g a n i c phosphate, s e v e r a l l i n e s of evidence showed t h a t the l i n k a g e was c o v a l e n t .  First,  the bound p h o s p h a t e - P  32  c o u l d not be  removed by passage of the l a b e l l e d protamine through Dowex 1 columns.  As shown i n F i g u r e  14, Dowex 1, a s t r o n g anion ex-  change r e s i n , q u a n t i t a t i v e l y r e t a i n e d i n o r g a n i c  phosphate-P  32  when i t was co-chromatographed w i t h u n l a b e l l e d protamine. The protamine, and s i m i l a r l y the l a b e l l e d phospho-protamine emerged from the column immediately a f t e r the v o i d w h i l e f r e e i n o r g a n i c phosphate-P. acid. P  3 2  all  32  volume,  was e l u t e d w i t h  Secondly, as i n d i c a t e d i n F i g u r e  hydrochloric  15, when phosphate-  l a b e l l e d protamine was adsorbed on carboxymethyl Sephadex, the protein-bound phosphate v/as r e t a i n e d on the column  when i t was e l u t e d w i t h water; the p h o s p h a t e - P  32  labelled  protamine was only e l u t e d subsequently w i t h d i l u t e a c i d . Under these c o n d i t i o n s  free inorganic phosphate-P  32  was not  r e t a i n e d on the column. The p u r i f i e d phospho-protamine was subjected phoresis  to electro-  i n a p o l y a c r y l a m i d e g e l a t pH 4.5. Autoradiography  of the g e l s t a i n e d w i t h Amido Black showed exact c o r r e s p o n dence between p r o t e i n and r a d i o a c t i v i t y i n the g e l (Figure 16), whereas opposite  free inorganic phosphate-P  32  m i g r a t e d i n the  d i r e c t i o n t o protamine, toward the anode.  It  appeared t h e r e f o r e t h a t t h i s phospho-protamine l i n k a g e was a covalent  one and f u r t h e r c h a r a c t e r i z a t i o n of the phospho-  -  86 -  • 1000 ^  0  20  40 60 80 Elution Volume,.. ml ^0 *«—- o. I M HCI  H  100 — *  F i g u r e 14„- Chromatography of P - l a b e l l e d 3 2  protamine  (A) and an admixture of u n l a b l l e d  protamine and f r e e i n o r g a n i c on a column of Dowex 1 x 8 , H0 2  and then 0.1 M HCI.  counts per minute  P . 3 2  •  phosphate-P e l u t i n g with •  A230,  o  32  (B)  first o  - 87 -  AMI DO BLACK STAINING  H  800 _cp D _  E  ro CO  o o  600  Protamine-P  /  400  free P  32  32  fx  CD  % i t \  200  X X X  O  0 &~=-8-  0  V.  20  H0 2  40 60 Elution Volume, ml  100  80  0.1 M HCI  F i g u r e 15 - Chromatography o f a mixture of P protamine and f r e e i n o r g a n i c p h o s p h a t e - P dex HCI. and  (H  +  32  3  -labelled  on CM Sepha-  form), e l u t i n g f i r s t with H 0, and then 0.1 M 2  A l i q u o t s of each f r a c t i o n were s p o t t e d on paper s t a i n e d with Amido Black 10B, the r e l a t i v e i n t e n s i t y  of t h i s s t a i n i n g , as shown i n t h i s f i g u r e , the p o s i t i o n of e l u t i o n o f protamine.  indicating  - 88 -  Polyacrylamide  Gel  Autoradiograph  B  Figure  B  16 - E l e c t r o p h o r e s i s  i n a polyacrylamide  of P - l a b e l l e d phospho-protamine 3 2  protamine from s t e e l h e a d  trout  gel  (A) and u n l a b e l l e d  (B), and  graphy of t h i s same g e l (A', B ' ) .  1  autoradio-  - 89  protamine was The age  initiated.  s t a b i l i t y of t h i s c o v a l e n t  to a c i d and  a l k a l i was  the p u r i f i e d p h o s p h a t e - P 15 minutes i n 0.1 droxide.  The  paper at pH  32  phospho-protamine  examined by h e a t i n g  subsequent high v o l t a g e  6.5  (Figure 17)  a l i q u o t s of  0.1  M sodium  electrophoresis  hyon  i n d i c a t e d t h a t the phospho-  s t a b l e to a c i d , but  l a b i l e to  r a d i o a c t i v i t y i n the a c i d - t r e a t e d sample had  ward the n e g a t i v e  link-  l a b e l l e d protamine at 100°C f o r  M h y d r o c h l o r i c a c i d and  p r o t e i n l i n k a g e was The  -  e l e c t r o d e as had  alkali. migrated t o -  protamine, w h i l e the  a c t i v i t y i n the a l k a l i - t r e a t e d sample migrated toward anode, the protamine again  toward the cathode.  This  the suggested  the presence of the s e r i n e e s t e r o-phosphoserine as had found i n the h i s t o n e s and  as r e p o r t e d  of salmine  (180)  of c a l f thymus  (75)  and  ( using  h y d r o c h l o r i c a c i d , 10 hours at 105°C) of a p u r i f i e d peptide  (Pi) from phospho-protamine.  (76),  hydrolysates  l a t e r found i n low  as a product of the p a r t i a l a c i d h y d r o l y s i s  been  r a t thymus  by Murray i n the p a r t i a l a c i d o-phosphoserine was  radio-  Ninhydrin  2.0  yield M  tryptic  positive  m a t e r i a l w i t h the same m o b i l i t y on e l e c t r o p h o r e s i s a t pH as known marker of o-phosphoserine was At t h i s pH,  o-phosphoserine was  still  obtained  (Figure  a n i o n i c w h i l e the  amino a c i d s of protamine as w e l l as glutamic  and  1.9 18). other  aspartic  a c i d were c a t i o n i c and migrated i n the other d i r e c t i o n .  The  low y i e l d of o-phosphoserine a f t e r p a r t i a l a c i d h y d r o l y s i s was  c o n s i s t e n t with the e a r l y c h a r a c t e r i z a t i o n of  - 90 -  Protamine  Electrophoretogram  0  Autoradiograph F i g u r e 17 - S t a b i l i t y of the c o v a l e n t phospho-protamine l i n k a g e t o a c i d and a l k a l i .  F o l l o w i n g treatment  of r e -  l a b e l l e d protamine w i t h 0.1 M HCI (1) or 0.1 M NaOH (2) samples were n e u t r a l i z e d and a p p l i e d t o Whatman 3 MM paper along with a marker o f f r e e i n o r g a n i c p h o s p h a t e - P (3) f o r e l e c t r o p h o r e s i s a t pH 6.5, 62 v o l t s / c m f o r 20 minutes.  A f t e r o b t a i n i n g the a u t o r a d i o g r a p h , the d r i e d  electrophoretogram was s t a i n e d w i t h Amido Black 10B.  32  - 91 -  •  • t  0  0 0  X  arginine  X  valine  X  serine  - 0  PI h y d r o l y s a t e  >  •  o-phosphoserine  0  +  F i g u r e 18 - N i n h y d r i n s t a i n e d electrophoretogram p a r t i a l acid hydrolysate  (2 M HCI,  of a  10 hours a t 105°C)  of the p u r i f i e d t r y p t i c phospho-peptide PI  (see  f o l l o w i n g s e c t i o n ) , along w i t h a p p r o p r i a t e amino a c i d markers. 3 MM  E l e c t r o p h o r e s i s was  paper a t pH  1.9,  c a r r i e d out on Whatman  62 v o l t s / c m f o r 20 minutes.  - 92 t h i s amino a c i d  (185) and the more r e c e n t work of Langan (81).  In those s t u d i e s r e c o v e r i e s of o-phosphoserine a f t e r  partial  a c i d h y d r o l y s i s have v a r i e d from 62 percent t o o n l y 25 percent.  In a f u r t h e r attempt t o demonstrate the presence o f  o-phosphoserine,  a t r y p t i c d i g e s t of a p h o s p h a t e - P  32  labelled  protamine p r e p a r a t i o n was s u b j e c t e d t o d i g e s t i o n w i t h the enzyme pronase (Calbiochem.) by adding  1.0 mg d i r e c t l y t o the  t r y p t i c d i g e s t i n 0.1 M ammonium b i c a r b o n a t e . free phosphate-P  32  However no  l a b e l l e d o-phosphoserine was o b t a i n e d ; the  enzyme pronase d i d not f u r t h e r d i g e s t the p e p t i d e s  obtained  with t r y p t i c d i g e s t i o n . (c) S i t e of P h o s p h o r y l a t i o n i n Protamine I t was of c o n s i d e r a b l e i n t e r e s t t o determine whether a s i n g l e unique s e r i n e i n protamine was phosphorylated, or whether p h o s p h o r y l a t i o n of s e r y l r e s i d u e s was a t a number of d i f f e r e n t s i t e s i n the protamine. such as phosphoglucomutase phosphatases  Phosphorylated  (186), hexokinase  proteins  (187) and s e v e r a l  (18 8, 189) are known t o be i n t e r m e d i a t e s i n  enzymic r e a c t i o n s , t h i s t r a n s i t o r y p h o s p h o r y l a t i o n o c c u r r i n g a t a s i n g l e s i t e , the " a c t i v e c e n t r e " i n each enzyme s u b u n i t . The  l i m i t e d p h o s p h o r y l a t i o n of the enzymes phosphcrylase  (190)  a  and UDPa-glucan t r a n s g l u c o s y l a s e (191) a t c e r t a i n .  d e f i n e d s i t e s has been shown t o be important t h e i r enzymatic  activity.  and milk phospho-proteins,  i n c o n t r o l of  In c o n t r a s t , the c l a s s i c egg y o l k vitellic  a c i d or p h o s v i t i n (192),  and c a s e i n (193) c o n t a i n r a t h e r l a r g e r amounts of phosphos e r i n e , as does a f u r t h e r c l a s s of phosphorylated p r o t e i n  - 93 r e c e n t l y demonstrated i n r a t l i v e r n u c l e i (81). To determine whether more than a s i n g l e s e r i n e i n protamine was  p h o s p h o r y l a t e d , the p u r i f i e d p h o s p h a t e - P  protamine was  digested with t r y p s i n .  has a s p e c i f i c i t y u n i q u e l y  32  labelled  T h i s p r o t e o l y t i c enzyme  s u i t e d f o r d i g e s t i o n of protamine,  as i t leads t o h y d r o l y s i s of the p e p t i d e bonds i n p r o t e i n s i n v o l v i n g the c a r b o n y l group of both a r g i n i n e and l y s i n e (194).  D i g e s t i o n of r a d i o a c t i v e protamine r e s u l t e d i n the  production  of r a d i o a c t i v e phospho-peptides which c o u l d be  e a s i l y f o l l o w e d d u r i n g the subsequent p u r i f i c a t i o n steps by the presence of t h i s  radioactivity.  The phospho-peptides were i s o l a t e d i n i t i a l l y  by  high  v o l t a g e e l e c t r o p h o r e s i s and descending chromatography  on  paper f o l l o w i n g the t r y p t i c d i g e s t i o n of the p u r i f i e d  phos-  phate-P  32  l a b e l l e d protamine.  T h i s p u r i f i c a t i o n has been des-  c r i b e d i n the M a t e r i a l s and Methods s e c t i o n shown d i a g r a m a t i c a l l y i n F i g u r e 19. covery  Because of the low r e -  of p e p t i d e m a t e r i a l from paper, i n subsequent e x p e r i -  ments the t r y p t i c p e p t i d e s chromatography is  ( c ) , and i s  on Dowex 50 x 2, and a t y p i c a l e l u t i o n diagram  shown i n F i g u r e 20.  organic phosphate-P , 32  (DI) ,. was  were f r a c t i o n a t e d by i o n exchange  A considerable  the f i r s t peak e l u t e d from the column  released during  the t r y p t i c d i g e s t i o n and subse-  quent work up.  T h i s h y d r o l y s i s of the  linkage during  the p r e p a r a t i o n o f the P  peptides  was  amount of f r e e i n -  phospho-protamine 3 2  labelled  tryptic  a problem not e n t i r e l y overcome i n t h i s work,  and both t h i s r e l e a s e of phosphate, and the low recovery  of  Tryptic Digest 32 of Protamine-P no  +  Electrophoresis at pH 6.5  P3  P2 C  ••>  -••  \ — \  Descending  PI  ,  f  i  4 Butanol/Formic  J  •  3  2  1  Electrophoresis at pH 1.9  Butanol/Acetic Chromatography  F i g u r e 19 - P u r i f i c a t i o n electrophoresis  of P - l a b e l l e d 3 2  t r y p t i c p e p t i d e s by  and chromatography on paper.  - 95 -  800 - 1 DI  UJ  _] a.600  5 <  5.0  _1 2  D I  CM  d  o: 400 UI a.  4.5  5  a. DU  200  4.0 £  D3ZE fi  A  ^D^Dm  200  3.5  400 600 ELUTION VOLUME ML  1800  ELUTION I WITH pH 5.0 J : BUFFER ~H  ELUTION WITH GRADIENT pH 3.1 TO 5.0  Figure  2 0 - Chromatography  of the t r y p t i c p e p t i d e s from r e -  l a b e l l e d protamine on Dowex 50 x 2; • P  3 2  per 0.2 ml sample,  i n the t e x t .  • counts per minute  pH o f e f f l u e n t .  peaks v/as pooled, l y o p h i l i z e d described  3.0 1000  1  Each o f the  and f u r t h e r c h a r a c t e r i z e d as  -  96  -  o-phosphoserine i n p a r t i a l a c i d h y d r o l y s a t e s of protamine, -may  i n d i c a t e a g r e a t e r l a b i l i t y than normal f o r o-phospho-  s e r i n e i n the b a s i c environment of the protamine c h a i n . The major r a d i o a c t i v e p e p t i d e o b t a i n e d by both paper, PI.,.- and column methods, D i l i , was  shown, by amino a c i d  a n a l y s i s and end group d e t e r m i n a t i o n , to be serine-arginine.  valine-phospho-  S e v e r a l other d i s t i n c t phosphoserine  pep-  t i d e s were o b t a i n e d as i n d i c a t e d i n Table IV, which summarizes  the a n a l y s i s of the major t r y p t i c  o b t a i n e d by both paper methods. one  I t was  (PI to P3)  phospho-peptides  and column  (DII to  DIV)  c l e a r t h a t the p h o s p h o r y l a t i o n of more than  s e r y l r e s i d u e had o c c u r r e d s i n c e f o u r d i f f e r e n t s e r i n e s  i n a t l e a s t t h r e e d i s t i n c t s e r i n e c o n t a i n i n g sequences were r e p r e s e n t e d i n the observed valine-serine-arginine arginine (P3).  (DII, DIV),  t r y p t i c phospho-peptides,  (Pi, Dili)  and  as above, s e r i n e - s e r i n e -  (alanine,serine,arginine), arginine  A complete d e t e r m i n a t i o n of the sequence  p e p t i d e , P3, was  of t h i s  last  not p o s s i b l e f o r i t had been i s o l a t e d i n  r a t h e r low y i e l d i n the paper p u r i f i c a t i o n method. t i d e , P2,  namely  The  pep-  ( v a l i n e , p h o s p h o s e r i n e , a r g i n i n e ) , a r g i n i n e , may  well  r e p r e s e n t the same sequence as the p e p t i d e PI, being a product of the incomplete DII and DIV may  In t h i s l a t t e r p e p t i d e , DIV,  were phosphorylated, s e r i n e s was  peptides  s i m i l a r l y r e p r e s e n t a s i n g l e sequence f o r  t h i s same reason.  two  d i g e s t i o n w i t h t r y p s i n , and  both s e r i n e s  w h i l e i n p e p t i d e DII only one  phosphorylated.  of the  F u r t h e r phospho-peptides  obtained on paper i n o n l y t r a c e amounts (Figure 19) c o u l d  -  97 -  Table IV A n a l y s i s o f Major T r y p t i c Phospho-Peptides Peptide  PI  P2  P3  DII  Dili  DIV  Composition Amino M i c r o A c i d * * moles Val  .026  Ser  .030  Arg  .031  Val  . 016  Ser  .017  Arg  .037  Ala  .008  Ser  .010  Arg  .022  Ser  .019  Arg  .011  Val  .034  Ser  .031  Arg  .034  Ser  .013  Arg  .012  N-Terminus  Sequence  Total Phosphorus  .030  P 1 VAL-SER-ARG§  Val*  P .023  |  -  (VAL,SER,ARG)ARG§ P  .013  -  .011  -  |  (ALA,SER,ARG)ARG§  f  (SER,SER)ARG P .034  Val*  VAL-SER-ARG§  p  .013  Argi  p  1 I ARG-SER-SER-ARG  P1-P3 -- Peptides i s o l a t e d by h i g h - v o l t a g e e l e c t r o p h o r e s i s and descending chromatography on paper (Figure 19). DII-DIV - Peptides i s o l a t e d by ion-exchange chromatography on Dowex 50 x 2 (Figure 20). *  I d e n t i f i e d as DNS-valine.  t  I d e n t i f i e d by s u b t r a c t i v e Edman procedure.  §  These s e r i n e sequences (nonphosphorylated) have a l s o been observed i n protamine from Clupea  pallasii  ** Values not c o r r e c t e d f o r l o s s e s d u r i n g  (8) .  acid hydrolysis.  hot be c h a r a c t e r i z e d ,  and  also represent  -  peaks DV,  -Dowex 50 -column (Figure 20) t o be impure and  98  DVI  and  DVII from  were shown by amino a c i d  to r e q u i r e f u r t h e r p u r i f i c a t i o n .  the analysis  They  may  the products of incomplete d i g e s t i o n w i t h  t r y p s i n f o r t h e i r p o s i t i o n of e l u t i o n from the column as  well  as the amino a c i d analyses i n d i c a t e d t h a t they were l a r g e r , more b a s i c  ( p o s i t i v e l y charged) p e p t i d e s than those o b t a i n e d  i n the peaks DII, As  D i l i and  DIV.  i n d i c a t e d i n Table IV,  which s e r i n e was  two  of the three  sequences i n  observed to be phosphorylated i n S.  gairdnerii  protamine were i d e n t i c a l to the sequences around s e r y l r e s i dues i n the components of protamine from Clupea The  t h i r d sequence, s e r i n e - s e r i n e - a r g i n i n e ,  these protamines from h e r r i n g although one YI, does c o n t a i n  i s not  (8).  found i n  component,  clupeine  a sequence s e r i n e - s e r i n e - s e r i n e - a r g i n i n e ,  does one  component of protamine from the P a c i f i c  0.  (19).  keta  pallasii  as  salmon,  (d) E x t e n t of P h o s p h o r y l a t i o n of Protamine S i n c e i t appeared l i k e l y t h a t s e r i n e r e s i d u e s  i n at  l e a s t f o u r d i s t i n c t p o s i t i o n s i n protamine c o u l d be phosphorylated,  i t was  phosphorylation  of i n t e r e s t to determine the e x t e n t of  of these s e r i n e s .  The  phosphorus, to t o t a l s e r i n e , which had  r a t i o of  been determined  amino a c i d a n a l y s i s of 16 hour h y d r o l y s a t e s tamine p r e p a r a t i o n s ,  was  as i n d i c a t e d i n Table V.  induced m a t u r a t i o n of the  by  of p u r i f i e d pro-  o b t a i n e d f o r a number of the  f e r e n t protamine p r e p a r a t i o n s stage of the  total  t e s t i s was  difThe  determined  - 99 Table V E x t e n t of P h o s p h o r y l a t i o n of Protamine a t D i f f e r e n t Stages o f Development Serine*  Source  Phosphorylation  Total P  Micromoles S.  (%)  gairdnerii  Testis (hormonally induced) f Early Intermediate  .061  .0454  74.4  .0585  .0115  20. 3  .123  .0113  9.2  .050  .0032  6.4  .087  0042  4.7  Late S.  gairdnerii  Mature  sperm  ( n a t u r a l spawning) 0.  kisutch  Mature  sperm  ( n a t u r a l spawning) * C o r r e c t e d f o r 6% l o s s o f s e r i n e during  acid  hydrolysis,  f Stage o f m a t u r a t i o n determined by v i s u a l i n s p e c t i o n o f excised  t e s t e s and p r o p o r t i o n s  i n a c i d e x t r a c t of t e s t e s .  of h i s t o n e  and protamine  - 100 by v i s u a l examination of the t e s t i s t i s s u e and by the proportions  of h i s t o n e  testes.  and protamine i n the a c i d e x t r a c t of the  A t l a t e stages of m a t u r a t i o n the t e s t i s t i s s u e was  l a r g e l y f r e e m i l t , and the protamine had almost e n t i r e l y replaced  histone  as the b a s i c p r o t e i n i n the t e s t i s  cell  nuclei. An  i n t e r e s t i n g r e l a t i o n s h i p between the stage of the  induced m a t u r a t i o n of the t e s t e s and the e x t e n t o f phosphoryl a t i o n o f s e r i n e i n protamine was immediately apparent.  Thus  i n the l e s s mature t i s s u e , the extent of p h o s p h o r y l a t i o n  of  s e r i n e was 74 p e r c e n t , w h i l e i n more mature t e s t e s t h i s phosphorylation  had dropped t o 20 p e r c e n t , and f i n a l l y t o  9 percent. The  p o s s i b i l i t y t h a t protamine might have s t i l l  sequences c o n t a i n i n g  s e r y l residues  which d i d not become  phosphorylated was next i n v e s t i g a t e d . mine l a b e l l e d w i t h the p r e c u r s o r r a t h e r than p h o s p h a t e - P  32  By p r e p a r i n g  amino a c i d  11  and c h a r a c t e r i z i n g the r a d i o a c t i v e serine-C *  l a b e l l e d protamine, the presence of f u r t h e r s e r i n e sequences might be demonstrated. 111  prota-  serine-C *  p e p t i d e s produced i n a t r y p t i c d i g e s t i o n of t h i s  with serine-C  further  11  containing  Protamine had been l a b e l l e d  i n a 2 hour i n c u b a t i o n  using  an a l i q u o t of the  same t e s t i s c e l l suspension w i t h which the i n c o r p o r a t i o n of inorganic  phosphate-P  32  had been s t u d i e d  p u r i f i c a t i o n of t h i s s e r i n e - C filtration Figure  1 k  (section(a)).  l a b e l l e d protamine by g e l  on Sephadex G-25 and B i o g e l P-10 i s shown i n  21., As w i t h p h o s p h a t e - P  The  32  labelled  preparations  ACID EXTRACTS ON G-25  RECHROMATOGRAPHY  ON BIOGEL P-IO  J  ELUTION V O L U M E , ML  F i g u r e 21 - G e l f i l t r a t i o n  chromatography of s e r i n e - C  the t e s t i s c e l l s  of S. g a i r d n e r i i  CFM s e r i n e - C .  Following  1 4  initial  1 4  l a b e l l e d p r o t e i n s e x t r a c t e d from  a f t e r incubation with s e r i n e - C . 1 4  chromatography on Sephadex G-25  •  • A220, o  the f r a c t i o n s  protamine were combined as i n d i c a t e d , l y o p h i l i z e d and rechromatographed on B i o g e l  o  containing P-10.  - 102  -  (Figure 13), both h i s t o n e and protamine peaks were seen to be l a b e l l e d w i t h r a d i o a c t i v e p r e c u r s o r , the protamine peak to a much g r e a t e r extent,  i n d i c a t i v e of the decrease i n h i s t o n e  s y n t h e s i s i n the t e s t i s c e l l p o p u l a t i o n at t h i s l a t e of maturation.  The  stage  protamine l a b e l l e d w i t h s e r i n e - C  was  1 4  i s o l a t e d i n pure s t a t e a f t e r rechromatography on B i o g e l A f t e r d i g e s t i o n of t h i s protamine w i t h t r y p s i n , first  step i n the p u r i f i c a t i o n procedure was  e l e c t r o p h o r e s i s on paper at pH  6.5.  y i e l d e d the same two  voltage  (Table V,  i n d i c a t e d t h a t o n l y 20 percent  s e r y l r e s i d u e s were phosphorylated,  the  Although the a n a l y s i s  of s e r i n e and phosphorus i n t h i s p r e p a r a t i o n mediate stage) had  high  P-10.  of  inter-  the  the t r y p t i c d i g e s t  p a r t i a l l y r e s o l v e d bands of  radioacti-  v i t y i n the n e u t r a l r e g i o n , as d i d the t r y p t i c d i g e s t of phosphate-P  (Figure 22, A, B).  The  s p e c i f i c i t y of t r y p s i n i s such t h a t i t should y i e l d  only  32  l a b e l l e d protamine  p e p t i d e s with a C - t e r m i n a l  arginine  these protamines - Table I ) .  (there i s no l y s i n e i n  As there are no a c i d i c amino  a c i d r e s i d u e s i n protamine other than phosphorylated the only n e u t r a l  peptides  peptides would c o n t a i n at l e a s t one be c a t i o n i c  serine,  t h a t c o u l d r e s u l t from t r y p t i c d i -  g e s t i o n are those which c o n t a i n o-phosphoserine.  minal) and  the  a r g i n y l residue  (basic) a t t h i s pH of 6.5.  p o s s i b i l i t y e x i s t e d , namely t h a t a p e p t i d e but no a r g i n i n e , hence n e u t r a l a t pH  A l l other  6.5,  from the C-terminus of whole protamine. of i n t a c t protamine w i t h carboxypeptidases  (C-terOne  other  containing serine c o u l d have come  However, d i g e s t i o n A and B had  shown  - 103 -  e ORIGIN  0 (A) Figure  (B)  22 - Autoradiographs o f h i g h - v o l t a g e  phoretograms o f t r y p t i c d i g e s t s of P protamine.  paper e l e c t r o -  - and s e r i n e - C  (A) i s the d i g e s t of p r o t a m i n e - P , 32  gest o f s e r i n e - C  1 4  1 4  labelled  (B) i s the d i -  protamine, and (C) shows the e f f e c t of  d e p h o s p h o r y l a t i o n of d i g e s t phatase.  3 2  (C)  (B) w i t h ff. c o l i  a l k a l i n e phos-  E l e c t r o p h o r e s i s was c a r r i e d out on Whatman 3 MM  paper,...pH 6.5, a t 62 volts/cm  f o r 20 min.  -  104  -  t h a t a r g i n i n e v/as the only C - t e r m i n a l  amino a c i d i n t h i s  -protamine (Figure 10), and hence even the p e p t i d e  released  from the C-terminus of protamine, would c o n t a i n a r g i n i n e . The  f i n d i n g t h a t only n e u t r a l r a d i o a c t i v e p e p t i d e s were  produced i n t h i s t r y p t i c d i g e s t i o n of the s e r i n e - C  protamine suggested t h a t a l l the newly i n c o r p o r a t e d r e s i d u e s were phosphorylated.  labelled  1 4  serine  T h i s i n t e r p r e t a t i o n was  sup-  p o r t e d by the o b s e r v a t i o n t h a t these n e u t r a l r a d i o a c t i v e peptides became b a s i c a f t e r treatment w i t h a l k a l i n e phosphatase.  The  serine-C  l a b e l l e d t r y p t i c p e p t i d e s were dephos-  1 4  p h o r y l a t e d by i n c u b a t i o n w i t h E. c o l i and  a l k a l i n e phosphatase  t h i s d i g e s t s u b j e c t e d t o e l e c t r o p h o r e s i s a t pH  w i t h the p h o s p h a t e - P tides.  32  and  serine-C  1 4  as  l a b e l l e d t r y p t i c pep-  A d i f f u s e area of r a d i o a c t i v i t y m i g r a t i n g much more  r a p i d l y toward the cathode i s seen i n F i g u r e 22 d i f f u s e appearance of t h i s band was to  6.5,  heavy o v e r l o a d i n g  t r y p t i c peptides  (C).  The  i n t e r p r e t e d as being  due  i n t h i s b a s i c r e g i o n where a l l other  from protamine  migrate.  Since newly s y n t h e s i z e d protamine appeared to have a l l its  s e r i n e r e s i d u e s phosphorylated  the extent of phosphory-  l a t i o n of protamine a t the other extreme, i n mature spermatozoa obtained  from f i s h a t the time of t h e i r spawning,  a l s o examined. gairdnerii  Protamine from the spermatozoa of both S.  (steelhead t r o u t ) and  p u r i f i e d as p r e v i o u s l y d e s c r i b e d t a i n only 4 to 6 percent phosphorylated  was  form  0. k i s u t c h  (coho salmon) were  (Part I) and  found to con-  of t h e i r s e r y l r e s i d u e s i n the  (Table V).  The  l a c k of p h o s p h o r y l a t i o n  of  -  105 -  t h i s m a t e r i a l i s i n agreement w i t h a l l p r e v i o u s protamines  work on  (4,5) i n which e i t h e r m i l t expressed from  fish,  or v e r y mature t e s t e s were used as s t a r t i n g m a t e r i a l i n the i s o l a t i o n and p u r i f i c a t i o n of protamine.  In these  earlier  s t u d i e s , the presence of o-phosphoserine was not noted. (e) K i n e t i c s of Protamine P h o s p h o r y l a t i o n I n h i b i t o r s of P r o t e i n The  and E f f e c t s of  Synthesis  suspension of c e l l s from the maturing t e s t e s of  S. g a i r d n e r i i preparation  (steelhead  t r o u t ) has been u t i l i z e d  f o r the  of amounts of phospho-protamine s u f f i c i e n t f o r  chemical c h a r a c t e r i z a t i o n of t h i s process of p r o t e i n phosphorylation. for smaller  These same c e l l  suspensions can be u t i l i z e d  s c a l e experiments s i m i l a r t o those d e s c r i b e d i n  P a r t I of t h i s t h e s i s . active precursor  J u s t as the i n c o r p o r a t i o n of a r a d i o -  amino a c i d i n t o protamine has d e l i n e a t e d  c e r t a i n f e a t u r e s of protamine b i o s y n t h e s i s , so s i m i l a r s t u d i e s of the i n c o r p o r a t i o n of i n o r g a n i c p h o s p h a t e - P  32  into prota-  mine can be employed t o e l u c i d a t e f e a t u r e s of the phosphoryl a t i o n of protamine. Protamine from the a c i d e x t r a c t of each i n c u b a t i o n was p u r i f i e d by e l e c t r o p h o r e s i s on p o l y a c r y l a m i d e  gels.  This  e l e c t r o p h o r e s i s was a p a r t i c u l a r l y e f f e c t i v e s t e p , f o r any inorganic phosphate-P  32  not c o v a l e n t l y bound t o protamine  migrated toward the o p p o s i t e  electrode.  The low background  at zero times i n t h i s i n c o r p o r a t i o n confirmed t h a t the complete separation  of f r e e and c o v a l e n t l y bound p h o s p h a t e - P  32  - 106 had  occurred.  The  c l e a r l y separated f o r as was  p r e p a r a t i v e e l e c t r o p h o r e s i s method a l s o h i s t o n e and protamine, an e s s e n t i a l f e a t u r e ,  seen i n F i g u r e 13, both h i s t o n e and protamine f r a c -  t i o n s are phosphorylated The P  3 2  -  i n these t e s t i s c e l l  preparations.  time course of i n c o r p o r a t i o n of i n o r g a n i c phosphate-  i n t o protamine i s shown i n F i g u r e 23.  The  phosphorylation  p a r a l l e l e d remarkably c l o s e l y the time course of i n c o r p o r a t i o n of a r g i n i n e - H  3  i n t o protamine i n the p r e v i o u s  studies  ( F i g u r e 5), a f i n d i n g c o n s i s t e n t w i t h the view t h a t r e s i d u e s i n newly s y n t h e s i z e d protamine were S i n c e the p h o s p h o r y l a t i o n  seryl  phosphorylated.  of protamine proceeded i n the -4  presence of puromycin a t a c o n c e n t r a t i o n of 10 as i n c o n t r o l i n c u b a t i o n s l a c k i n g puromycin p h o s p h o r y l a t i o n must be a process  ( F i g u r e 23),  this  independent of the i n c o r -  p o r a t i o n of amino a c i d s i n t o protamine, f o r the t i o n of puromycin i n these i n c u b a t i o n s was h i b i t the i n c o r p o r a t i o n of a r g i n i n e - H percent  M exactly  3  concentra-  s u f f i c i e n t to i n -  i n t o protamine  80  (Figure 6).  K l e i n s m i t h et al  (75) have a l s o shown t h a t the phosphory-  l a t i o n of n u c l e a r p r o t e i n s i s independent of p e p t i d e bond formation.  Under c o n d i t i o n s i n which puromycin markedly i n -  h i b i t e d the i n c o r p o r a t i o n of s e r i n e - C * i n t o the p r o t e i n s of 11  thymus n u c l e i , the p h o s p h o r y l a t i o n r e a c t i o n ted.  A s i m i l a r phosphorylation  little  of preformed p r o t e i n was  d i c a t e d i n the experiments of S t e v e l y and the p h o s p h o r y l a t i o n  was  Stocken  affecin-  (76), f o r  of the h i s t o n e f l f r a c t i o n of r a t thymus  n u c l e i v/as not i n h i b i t e d by puromycin at a c o n c e n t r a t i o n of  -  0  107  -  30  60  90  Time , minutes  F i g u r e 23 - Time course of i n c o r p o r a t i o n of i n o r g a n i c phate-P  32  i n t o protamine by a suspension of S.  (steelhead t r o u t ) t e s t i s  cells.  phos-  gairdnerii  - 108 100  ygm  per ml.  -  I t would appear t h a t the p h o s p h o r y l a t i o n  these p r o t e i n s resembles c e r t a i n other  r e a c t i o n s i n which  primary s t r u c t u r e of p r o t e i n s i s m o d i f i e d . and m e t h y l a t i o n hemoglobin  of h i s t o n e s  (197)  (74,196) and  The  t i o n of a p r e v i o u s l y e x i s t i n g p o l y p e p t i d e  the  acetylation  the a c e t y l a t i o n of  appear to be cases i n which the  modifica-  chain-is  by s p e c i f i c enzymatic t r a n s f e r r e a c t i o n s . phospho-kinase enzyme has  of  The  catalyzed  presence of a  been demonstrated r e c e n t l y i n r a t  l i v e r t i s s u e (198)  as w e l l as i n the t e s t i s t i s s u e of S.  gairdnerii  These enzymes r e s u l t i n the phosphoryla-  (199) .  t i o n of h i s t o n e u t i l i z i n g ATP  f r a c t i o n s (198)  and  protamine  (198,199),  as phosphate donor.  A s i m i l a r examination of the mechanism by v/hich phosphorus i s i n c o r p o r a t e d gland  has  been r e p o r t e d  i n t o c a s e i n molecules i n the mammary (200).  Phosphorus might be  duced e i t h e r i n the preformed p o l y p e p t i d e amino a c i d l e v e l ,  chains  intro-  or a t  i . e . s e r i n e c o u l d be p h o s p h o r y l a t e d  the o-phosphoserine thus formed might subsequently be  the and incor-  porated  i n t o the c a s e i n molecule by means of a s p e c i f i c  f e r RNA  (tRNA) f o r o-phosphoserine.  c o u l d occur w h i l e the p o l y p e p t i d e  The  trans-  former mechanism  chains were s t i l l  attached  to ribosomes, as has  been i n d i c a t e d f o r the i n c o r p o r a t i o n of  glucosamine r e s i d u e s  i n t o the g l y c o p r o t e i n s  tissue somes.  of r a t  liver  (201), or a l t e r n a t i v e l y , a f t e r t h e i r r e l e a s e from r i b o The  phosphate-P  study of the i n v i t r o 32  i n c o r p o r a t i o n of  inorganic  i n t o c a s e i n i n the presence of puromycin as a  f u n c t i o n of time r e v e a l e d  t h a t puromycin d i d not  appreciably  - 109 a f f e c t phosphate-P  32  i n c o r p o r a t i o n d u r i n g a 30 minute  t i o n , but d i d i n h i b i t a f t e r longer time i n t e r v a l s . t h e s i s of new p r o t e i n was b l o c k e d and  immediately  with  incuba-  The synpuromycin,  t h e r e f o r e t h i s r e s u l t i n d i c a t e d t h a t a p o o l o f non-  phosphorylated  or i n c o m p l e t e l y phosphorylated  A t longer i n c u b a t i o n times  casein existed.  the a v a i l a b i l i t y of non-phosphory-  l a t e d p r e c u r s o r became the l i m i t i n g f a c t o r and p h o s p h a t e - P i n c o r p o r a t i o n was then  32  inhibited.  As the p r e v i o u s study of s e r i n e - C * i n c o r p o r a t i o n i n t o 11  the phospho-peptides of protamine had i n d i c a t e d t h a t a l l newly i n c o r p o r a t e d s e r y l r e s i d u e s were phosphorylated f i n d i n g t h a t puromycin d i d not i n h i b i t t h i s  the  phosphorylation  r e a c t i o n i n a manner s i m i l a r t o i t s i n h i b i t i o n of phosphoryl a t i o n of c a s e i n even a f t e r 9 0 minutes, was somewhat unexpected.  That the s i t u a t i o n was even more complex became  apparent i n a f u r t h e r experiment i n which the t e s t i s were incubated  i n the presence of c y c l o h e x i m i d e .  This  b i t o r a t a c o n c e n t r a t i o n of 5 x 10 ^ M had been shown ure 6) t o completely H  3  cells inhi(Fig-  i n h i b i t the i n c o r p o r a t i o n of a r g i n i n e -  i n t o protamine, and t h e r e f o r e i t was expected  i n a s i g n i f i c a n t decrease  i n protamine  to r e s u l t  phosphorylation.  However as shown i n Table V I , r a t h e r than i n h i b i t the phos-: p h o r y l a t i o n , the cycloheximide a t i o n of p h o s p h a t e - P  32  l e d t o an i n c r e a s e d i n c o r p o r -  i n t o protamine.  T h i s s t i m u l a t i o n of  p h o s p h o r y l a t i o n became p a r t i c u l a r l y marked a t the longer times of i n c u b a t i o n .  -  110 -  Table V i : E f f e c t of Cycloheximide  on Protamine P h o s p h o r y l a t i o n  Time of I n c u b a t i o n (minutes)  Expt. No. 1  2  Incorporation P as percent of c o n t r o l 3 2  20  107  40  152  90  186  45  136  90  183  An e x p l a n a t i o n f o r these r e s u l t s w i t h i n h i b i t o r s of p r o t e i n s y n t h e s i s i s not immediately  apparent.  p l e t e c e s s a t i o n of protamine s y n t h e s i s w i t h  The com-  cycloheximide  has r e s u l t e d i n an i n c r e a s e i n protamine p h o s p h o r y l a t i o n w h i l e an 80 percent i n h i b i t i o n of protamine s y n t h e s i s w i t h puromycin d i d not r e s u l t i n any for this The  i n c r e a s e over c o n t r o l values  phosphorylation. l a c k of any  i n h i b i t i o n of the p h o s p h o r y l a t i o n of  protamine by these two puromycin and  i n h i b i t o r s of p r o t e i n s y n t h e s i s ,  cycloheximide,  suggests  s u f f i c i e n t supply of dephosphorylated  t h a t there must be a protamine which  serve as s u b s t r a t e i n the p h o s p h o r y l a t i o n r e a c t i o n . v i o u s experiments had protamine was  can The  :  pre-  i n d i c a t e d t h a t the p h o s p h o r y l a t i o n of  a t l e a s t i n p a r t coupled to i t s s y n t h e s i s , f o r  a l l newly i n c o r p o r a t e d s e r y l r e s i d u e s i n protamine were to be phosphorylated.  However t h i s p h o s p h o r y l a t i o n  found  continues,  - Ill and  -  i n the case of cycloheximide, i s even s t i m u l a t e d i n the  absence of an a p p r e c i a b l e amount of new I t i s apparent protamine,  protamine  t h e r e f o r e t h a t a supply of  synthesis.  dephosphorylated  a p a r t from t h i s p o o l of newly s y n t h e s i z e d  protamine,  must e x i s t . . The  two  i n h i b i t o r s , puromycin and c y c l o h e x i m i d e ,  may  a c t i n these t e s t i s c e l l s not only by i n h i b i t i n g the f o r m a t i o n of new  protamine,  but a l s o by i n t e r r u p t i n g the t r a n s p o r t of  newly synthesized, protamine ribosomes  from i t s s i t e of s y n t h e s i s on  (202,203) t o the chromatin.  s i s of other p r o t e i n s may replacement  The  continued  synthe-  be a p r e r e q u i s i t e f o r the o r d e r l y  of h i s t o n e s i n the chromatin by protamine,  and  the i n t e r r u p t i o n of t h i s process might y i e l d a p o o l of  de-  phosphorylated protamine The  which c o u l d be  rephosphorylated.  turnover of these phosphate groups i n the presence  phosphate-P  32  c o u l d t h e r e f o r e g i v e r i s e t o the h i g h e r  f i c a c t i v i t i e s of the p h o s p h a t e - P i n these  32  l a b e l l e d protamine  of speciobserved  experiments.  Both puromycin and cycloheximide may p r o t e i n s y n t h e s i s i n the t e s t i s c e l l s , utilization,  and w i t h i n c r e a s e d ATP  the p h o s p h o r y l a t i o n of protamine d e p h o s p h o r y l a t i o n r e a c t i o n may i n i n c r e a s e d protamine  a l s o , by  inhibiting  lead to reduced  l e v e l s i n these  cells,  c o u l d be s t i m u l a t e d .  a l s o be i n h i b i t e d ,  ATP  The  resulting  phosphorylation; for i t i s l i k e l y that  t h i s d e p h o s p h o r y l a t i o n i s c a r r i e d out by s p e c i f i c phosphatase enzymes, and  the continued s y n t h e s i s of these enzymes may  necessary t o ensure adequate d e p h o s p h o r y l a t i o n of  be  protamine.  - 112 The  s y n t h e s i s of protamine  on ribosomes  (202,203) and  i t s subsequent t r a n s p o r t t o the chromatin where i t binds w i t h DNA i s l i t t l e understood.  W i t h . t h i s process of s y n t h e s i s and  t r a n s p o r t , t h e r e i s a p a r a l l e l p h o s p h o r y l a t i o n and e v e n t u a l d e p h o s p h o r y l a t i o n of protamine. of  these experiments  A s a t i s f a c t o r y explanation  w i t h puromycin and c y c l o h e x i m i d e must  await a more complete understanding protamine  of the e n t i r e process of  s y n t h e s i s and the replacement  t i n by t h i s  of h i s t o n e i n chroma-  protamine.  POSSIBLE BIOLOGICAL ROLE of PROTAMINE PHOSPHORYLATION I t has been w i d e l y suggested the c e l l (65,74).  t h a t the b a s i c p r o t e i n s of  n u c l e i a s s i s t i n the r e g u l a t i o n of gene e x p r e s s i o n Stedman and Stedman f i r s t proposed  t h a t these b a s i c  p r o t e i n s of the c e l l n u c l e i were gene i n h i b i t o r s , each d i f f e r e n t h i s t o n e or protamine  being capable of s u p p r e s s i n g the  a c t i v i t y of s p e c i f i c groups o f genes of  (63,64).  The process  c e l l d i f f e r e n t i a t i o n i m p l i e d t h a t d i f f e r e n t groups of  genes were a c t i v e i n d i f f e r e n t c e l l s and these felt  investigators  t h a t t h e i r hypothesis gave some i n d i c a t i o n of the mechan-  ism f o r a d i f f e r e n t i a l e x p r e s s i o n of those n u c l e a r genes. However attempts of  t o d i s c o v e r c o r r e l a t i o n s between the s t a t e  d i f f e r e n t i a t i o n of t i s s u e and q u a l i t a t i v e d i f f e r e n c e s i n  i t s h i s t o n e content have l a r g e l y been u n s u c c e s s f u l (66) .  In  g e n e r a l , h i s t o n e s from a wide v a r i e t y of sources have proved to  have much i n common.  A comparison of h i s t o n e s prepared  from the p u r i f i e d chromatin  from buds of pea s e e d l i n g s , and  - 113  -  from c a l f thymus has been r e p o r t e d  (72).  The  h i s t o n e s of  these very d i s t a n t l y r e l a t e d organisms were remarkably s i m i l a r , both upon f r a c t i o n a t i o n by i o n exchange chromatography and  electrophoresis i n polyacrylamide  amino a c i d composition  and N-terminal  various histone f r a c t i o n s .  g e l s , as w e l l as i n the amino a c i d s of  In a f u r t h e r r e p o r t the  the histones  of p u r i f i e d chromatin from a number of d i f f e r e n t t i s s u e s of the pea p l a n t were compared  (73).  p r e s e n t were s p e c i f i c to any  None of the  histones  pea t i s s u e , although  a difference  i n the q u a n t i t a t i v e d i s t r i b u t i o n of c e r t a i n of these f r a c t i o n s i n d i f f e r e n t t i s s u e s was The  histone  noted.  o r i g i n a l concept t h a t d i f f e r e n t h i s t o n e s might  found i n d i f f e r e n t types  of c e l l s from one  organism  from the o b s e r v a t i o n t h a t the spermatozoa of f i s h  be  arose  contained  the unusual b a s i c p r o t e i n , protamine, which d i f f e r e d markedly from the b a s i c p r o t e i n s found i n somatic t i s s u e s of the same animal.  A f u r t h e r example of a t i s s u e s p e c i f i c i t y f o r h i s -  tones was  demonstrated i n the a v i a n e r y t h r o c y t e  major h i s t o n e f r a c t i o n r i c h i n both l y s i n e and w e l l as s e r i n e was  p e c u l i a r to these c e l l s ,  (204), and  a  a r g i n i n e as  apparently  re-  p l a c i n g the " a r g i n i n e - r i c h " h i s t o n e of other somatic t i s s u e s (205).'  Apart  from these  two  c l e a r exceptions,  f i n e d methods of h i s t o n e a n a l y s i s have f a i l e d  the more r e to demonstrate  the e x i s t e n c e of markedly d i f f e r e n t h i s t o n e s i n d i f f e r e n t t i s s u e s and  among d i f f e r e n t s p e c i e s  (66,72,73).  The  occur-  rence of t i s s u e s p e c i f i c b a s i c p r o t e i n s i n both spermatozoa and a v i a n e r y t h r o c y t e s may  be r e l a t e d to the l i m i t e d b i o -  - 114 s y n t h e t i c c a p a c i t i e s of these  -  two  types of c e l l s .  Both  spermatozoa and a v i a n e r y t h r o c y t e s are c h a r a c t e r i z e d by t h e i r s m a l l condensed n u c l e i , the l a c k of f u r t h e r c e l l s i o n , and  c e s s a t i o n of RNA  t h a t protamine, and may  synthesis  (86,87).  divi-  It i s possible  s i m i l a r l y the a v i a n e r y t h r o c y t e  histone  p l a y a r o l e i n the g e n e r a l i z e d r e p r e s s i o n of gene a c t i v i t y  i n these The  two  types  of h i g h l y s p e c i a l i z e d  cells.  p o s s i b i l i t y t h a t the g e n e t i c r e g u l a t i o n might be a t  a more s u b t l e l e v e l of h i s t o n e chemistry  became apparent  when a s e r i e s of r e a c t i o n s were observed i n which h i s t o n e s were c h e m i c a l l y m o d i f i e d by e i t h e r a c e t y l a t i o n (74) methylation  (74), or p h o s p h o r y l a t i o n  (75-77) .  and  These r e a c -  t i o n s have been proposed as an a l t e r n a t i v e mechanism to o b t a i n s p e c i f i c r e g u l a t o r e f f e c t s on the DNA both a c e t y l a t i o n (74)  and  phosphorylation  template.  (76) of the  have been shown to have marked e f f e c t s on the of the DNA  template i n t o RNA  in v i t r o .  Complexes of DNA  Indeed histones  transcription  i n s t u d i e s with RNA  polymerase  w i t h e i t h e r a c e t y l a t e d or phos-  p h o r y l a t e d h i s t o n e s showed an i n c r e a s e d a b i l i t y to prime s y n t h e s i s compared to s i m i l a r complexes of DNA a c e t y l a t e d , or non-phosphorylated h i s t o n e s . i n c r e a s e i n a c e t y l a t i o n (74)  and  with  (75)  n u c l e a r p r o t e i n s c o r r e l a t e d with an i n c r e a s e of RNA i n lymphocytes s t i m u l a t e d to grow and glutinin  (a p r o t e i n d e r i v e d from kidney  be r e l a t e d to the a c t i v i t y of DNA  an  of  synthesis  d i v i d e by phytohaemagbeans).  I t would  appear then t h a t these more s u b t l e m o d i f i c a t i o n s of may  non-  Furthermore,  phosphorylation  RNA  histones  i n chromatin i n s e r v i n g  - 115 as a template  f o r RNA  synthesis.  The p h o s p h o r y l a t i o n Stocken  (76) was  -  of h i s t o n e s r e p o r t e d by S t e v e l y  of a r a t h e r l i m i t e d nature.  The  phosphorus  content of the h i s t o n e f l f r a c t i o n v a r i e d between 20 and mu  moles of phosphorus per mg  there was  o n l y 0.3  histone.  The  to 0.6  45  of p r o t e i n , i n d i c a t i n g t h a t  moles of phosphate per mole of  combined s e r i n e and  histone f l f r a c t i o n  and  t h r e o n i n e content of  this  (molecular weight about 14000) i s 10.5  moles per 100 moles of t o t a l amino a c i d s  (206) , thus only a  s m a l l p r o p o r t i o n of the p o t e n t i a l s i t e s f o r p h o s p h o r y l a t i o n were observed  to be  phosphorylated.  In the s t u d i e s of the p h o s p h o r y l a t i o n of the b a s i c prot e i n s of S. g a i v d n e v i i  t e s t i s c e l l s r e p o r t e d here,  but f a r more e x t e n s i v e p h o s p h o r y l a t i o n has been observed.  In the d e v e l o p i n g  a similar  of n u c l e a r p r o t e i n s testes, cells  undergoing  d i f f e r e n t i a t i o n to mature spermatozoa c o n t a i n protamine which was  found  to have up to 75 percent of i t s s e r y l r e s i d u e s phos-  phorylated.  The  turnover  of t h i s phosphate has been i n d i c a t e d  by the f i n d i n g t h a t i n newly s y n t h e s i z e d protamine a l l s e r y l r e s i d u e s were phosphorylated,  w h i l e i n mature spermatozoa  these same s e r y l r e s i d u e s appeared to be completely phorylated.  I t i s p o s s i b l e t h a t t h i s turnover of phosphate  e s t e r i f i e d to s e r i n e may l a t i o n of the template  p l a y an important  a c t i v i t y of DNA  the l a t e stages of spermatogenesis.  at the three  r o l e i n the  i n chromatin  regu-  during  For example, the  d u c t i o n of up to 6 or 8 n e g a t i v e charges pH)  dephos-  intro-  (at p h y s i o l o g i c a l  or f o u r s e r y l r e s i d u e s i n each protamine  -  116  -  c o u l d impose a c o n t r o l on e i t h e r the extent or nature of — c o m p l e x i n g of t h i s protamine w i t h DNA,  and  thus  the  i n f l u e n c e the  degree of gene e x p r e s s i o n . "The replacement of h i s t o n e s i n the t e s t e s by protamine may  a l s o be a process c o n t r o l l e d i n p a r t by the degree of  p h o s p h o r y l a t i o n of these b a s i c p r o t e i n s .  The  s p e c i f i c phos-  p h o r y l a t i o n of e i t h e r h i s t o n e or protamine a t the stage may  appropriate  f a c i l i t a t e the removal of h i s t o n e from i t s complex  i n chromatin protamine.  with DNA, Again  and  i t s subsequent replacement by  the a d d i t i o n of n e g a t i v e charges i n the  form of o-phosphoserine or o-phosphothreonine may  influence  the degree of complexing of e i t h e r h i s t o n e or protamine to DNA. Protamine, d e s p i t e i t s s m a l l s i z e and unusual composition  has a l r e a d y been shown to be s y n t h e s i z e d by a  system i n v o l v i n g mRNA, tRNA and ribosomes cellular  amino a c i d  (Part I ) .  The  sub-  l o c a l i z a t i o n of the s y n t h e s i s of both protamine  h i s t o n e s i s a s u b j e c t of some d i s p u t e at p r e s e n t .  Reid  and and  C o l e have shown the s y n t h e s i s of the w e l l c h a r a c t e r i z e d h i s t o n e f l f r a c t i o n i n i s o l a t e d c a l f thymus n u c l e i  (207).  In  other work, the s y n t h e s i s of such n u c l e a r p r o t e i n s has a l s o been judged to be n u c l e a r added r i b o n u c l e a s e and sium i o n s . t h a t nascent  (208,209) f o r i t was  resistant  dependent on sodium r a t h e r than  On the other hand, Robbins and Borun h i s t o n e s i n synchronized  (210)  to  potasfound  p o p u l a t i o n s of Hela  c e l l s were l o c a t e d on s m a l l polyribosomes  i n the  cytoplasm.  T h i s l a t t e r o b s e r v a t i o n i s i n accord with the s t u d i e s of  - 117 -  Block and Brack (134).  Using autoradiographic techniques,  the "arginine-rich" histones of the grasshopper vividifasoiata)  {Chortophagia  spermatids appeared to be present f i r s t i n  the cytoplasm, and only l a t e r i n the nucleus of these c e l l s . U t i l i z i n g t e s t i s c e l l s at a stage active i n the synthesis of protamine, pulse-chase experiments with a r g i n i n e - C  14  have  shown the s i t e of synthesis of protamine to be on cytoplasmic microsomes associated with, but e a s i l y detached from the nucleus  (202,203).  The c h a r a c t e r i s t i c properties of protamine as a p r e c i p i t a t i n g agent for nucleic acids (211), raises the question of how a polycationic protein such as protamine i s synthesized on ribosomes i n the presence of mRNA and tRNA without causing p r e c i p i t a t i o n of these molecules and aggregation of the synt h e t i c machinery.  A number of effects of histones on membrane  function have also been noted (212), and therefore the synthesis of these nuclear proteins at any subcellular s i t e removed from the chromatin immediately leads to the problem of the transportation of these proteins within the c e l l to t h e i r f i n a l location i n the chromatin.  The phosphorylation of basic pro-  teins at the time of their synthesis may considerably modify their tendency to complex with RNA or i n t e r f e r e with membrane function.  This phosphorylation might then f a c i l i t a t e trans-  port of the newly synthesized proteins from the s i t e of synthesis to their destination i n the chromatin.  A  subsequent  dephosphorylation could i n i t a t e t i g h t binding to the  DNA.  This sequence of events i s shown i n a schematic representation  - 118 (Figure 24).  -  Protamine, phosphorylated  by a kinase enzyme  when newly s y n t h e s i z e d , i s t r a n s p o r t e d to the where dephosphorylation of protamine to  chromatin  i s shown as i n i t i a t i n g the  binding  DNA.  Although the evidence  presented  here i n d i c a t e d t h a t  a l l newly i n c o r p o r a t e d s e r y l r e s i d u e s i n protamine were phosphorylated,  a s i m i l a r phosphorylation  h i s t o n e s has y e t to be demonstrated. composition  of newly The  synthesized  unique amino a c i d  of protamine, i n p a r t i c u l a r the absence of  a c i d i c amino a c i d r e s i d u e s p e r m i t t e d  an examination of  newly s y n t h e s i z e d protamine molecules and phosphorylation.  (214),  phospho-peptides  for purification  suggests a manner i n which the  s y n t h e s i s of h i s t o n e s and a concomitant might be examined.  their  (213), a f u r t h e r a p p l i c a t i o n  of the d i a g o n a l e l e c t r o p h o r e t i c technique of s p e c i f i c peptides  the  the extent of  The method f o r i s o l a t i n g  r e c e n t l y r e p o r t e d by M i l s t e i n  any  phosphorylation  A s h o r t time i n c u b a t i o n with  f o l l o w e d by p u r i f i c a t i o n of the h i s t o n e s and  the  serine-C "* 1  subsequent  i s o l a t i o n of the phospho-peptides would answer whether a l l the newly i n c o r p o r a t e d and hence r a d i o a c t i v e s e r y l were phosphorylated.  The  low amount of phosphate i n the  i s o l a t e d h i s t o n e f r a c t i o n s r e p o r t e d by S t e v e l y and (76), may  be o n l y an i n d i c a t i o n of the r e l a t i v e l y  amount of new  residues  Stocken small  s y n t h e s i s of these p r o t e i n s i n progress  at  the same time of t h e i r i s o l a t i o n from the l i v e r t i s s u e . t h i s regard  i t i s of i n t e r e s t t h a t a two  the l e v e l of phosphate i n h i s t o n e occurs  In  f o l d increase i n 16 to 24 hours a f t e r  -119  -  F i g u r e 2 4 - Schematic r e p r e s e n t a t i o n of the events d u r i n g synthesis, phosphorylation the chromatin.  and  t r a n s p o r t of protamine to  Protamine, phosphorylated  by a k i n a s e when  newly s y n t h e s i z e d on ribosomes, i s t r a n s p o r t e d to  the  chromatin where i t r e p l a c e s h i s t o n e s i n b i n d i n g to the DNA.  Dephosphorylation  at t h i s point could i n i t i a t e  t i g h t b i n d i n g of protamine to  DNA.  the  - 120 p a r t i a l hepatectomy  (215).  T h i s i s a t the time when the  s y n t h e s i s of both h i s t o n e and DNA i s known t o occur In  (216).  a study of the enzymes i n v o l v e d i n t h i s process of  the p h o s p h o r y l a t i o n of protamine, phosphorylate protamine i n the high-speed  a phospho-kinase  has been demonstrated  (199).  supernatant of a tris-magnesium  e x t r a c t of S. g a i v d n e v i i  which does Present  chloride  t e s t i s t i s s u e , the enzyme has been  p a r t i a l l y p u r i f i e d by chromatography on Sephadex G-200 and DEAE c e l l u l o s e .  L i k e other k i n a s e s i t r e q u i r e s ATP as  phosphate donor, and the presence of magnesium i o n s and a thiol activator  (3-mercaptoethanol).  A s i m i l a r enzyme,  " h i s t o n e k i n a s e " , which phosphorylates both h i s t o n e s and protamine (198).  has been r e p o r t e d by Langan i n r a t l i v e r  In a d d i t i o n a phosphatase  protamine  specific  f o r h i s t o n e s and  has been i s o l a t e d from t h i s same t i s s u e  Thus an enzymatic i n protamine  tissue  (217).  b a s i s f o r the observed phosphate turnover  as w e l l as h i s t o n e s appears  t o have been  established. In  summary, P a r t I of t h i s t h e s i s was p r i m a r i l y  con-  cerned w i t h the b i o s y n t h e s i s and chemical c h a r a c t e r i z a t i o n of  protamine.  The s y n t h e s i s o f protamine  was s t u d i e d i n  both the t e s t i s t i s s u e of n a t u r a l l y maturing  salmonoid  fish  as w e l l as i n the t e s t i s t i s s u e of immature S. g a i v d n e v i i . Spermatogenesis  c o u l d be induced i n these l a t t e r f i s h by  i n j e c t i o n s o f p i t u i t a r y e x t r a c t s , and thus a convenient source o f t i s s u e f o r the experiments was p r o v i d e d .  Protamines  on protamine s y n t h e s i s  from s e v e r a l d i f f e r e n t s p e c i e s of  - 121 salmonoid f i s h were p u r i f i e d by a g e l f i l t r a t i o n method, and the amino a c i d compositions of them determined. t i o n of N and C - t e r m i n a l  Characteriza-  amino a c i d s as w e l l as attempts a t  f r a c t i o n a t i n g the components of protamine were a l s o i n Part I.  The p h o s p h o r y l a t i o n  of P a r t I I of t h i s t h e s i s .  described  of protamine was the s u b j e c t  The chemical nature of the phos-  p h o r y l a t i o n was i n v e s t i g a t e d , the s i t e s of. p h o s p h o r y l a t i o n protamine determined, and o v e r a l l c h a r a c t e r i s t i c s of t h i s phosphorylation  r e a c t i o n examined.  in  - 122 BIBLIOGRAPHY 1.  Miescher, F., D i e Histochemischen und P h y s i o l o g i s c h e n A r b e i t e n von F r i e d e r i c h Miescher, 2 v o l s . , F.C.W. V o g e l , L e i p z i g , 1897.  2.  K o s s e l , A. The Protamines  and H i s t o n e s , Ed. Longmans,  Green and Co., London, 1928. 3.  Murray, K. , Annual Rev. Biochem. 34_,  4.  F e l i x , F., Advances i n P r o t e i n Chemistry ]L5, 1, 1960.  5.  Vendrely, R. and C. Vendrely, P r o t o p l a s m a t o l o g i a _5, 3c S p r i n g e r - V e r l a g , Vienna, 1966. Ando, T., K. Iwai, S. I s h i i , M. Azeg:ami and C. Nakara, Biochim. Biophys. Acta 5_6, 628, 1962.  6.  2  0  9  / 1965.  7.  Ando, T. and K. Suzuki, Biochim. Biophys. A c t a 121, 1966.  8.  Ando, T., K. Suzuki, S. Watenabe and S. Inoue, V I I I n t e r nat. Congr. Biochem., Tokyo, ( a b s t r a c t ) , 1967.  9.  Daly, M.M., A.E. Mirsky and H. R i s , J . Gen. P h y s i o l . 34, 439, 1951.  10.  F i s c h e r , H. and L. Kreuzer, Z. P h y s i o l . Chem. 239, 1953.  11. I B r i l - P e t e r s e n , E. and H.G.K. Westenbrink,  Biochim.  427,  176, Biophys.  A c t a 7J5, 152, 1963. 12.  F e l i x , K. and K. D i r r ,  Z. P h y s i o l . Chem. 184, 111, 1929.  13. 14.  F e l i x , K., C i b a Found. Symp., C h u r c h i l l , London, p. 151, 1953. Scanes, F.S. and B.T. Tozer, Biochem. J . 6_3, 565, 1956.  15.  Ando, T., S. I s h i i , M. Yamasaki, K. Iwai, C. Hashimoto and F. Sawada, J . Biochem. (Tokyo) £ 4 , 275, 1957.  16.  Ando, T. and F. Sawada, J . Biochem. (Tokyo) £6, 517,  17.  Ando, T. and F. Sawada, J . Biochem. (Tokyo) £ 9 , :252, 1961.  18.  Ando, T. and K. S u z u k i , Biochim. Biophys. A c t a 140, 1967. Ando, T., p e r s o n a l communication.  19.  1959.  375,  - 123 20.  W i l k i n s , M.H.F., Cold Sp. Harb. Symp. Quant. B i o l . 1956.  21.'  21.  B l a c k , J.A. and G.H. Dixon, Nature 216, 152, 1967.  22.  S m i t h i e s , 0., G.E. C o n n e l l and G.H. Dixon, Nature 196,  75,  232, 1962. 23.  Dixon, G.H. , Essays i n B i o c h e m i s t r y 2_,  24.  F e l i x , K., A. Goppold-Krekels and H. Lehmann, Z. P h y s i o l . Chem. 312, 57, 1958. Ando, T. and C. Hashimoto, J . Biochem. (Tokyo) 45, 529,  25.  147, 1966.  1958. 26.  A l f e r t , M., J . Biophys. Biochem. C y t o l . 2, 109, 1956.  27.  B l o c h , D.P., J . Histochem. Cytochem.  28.  W i l s o n , E.B., The C e l l i n Development and H e r e d i t y , 3rd e d i t i o n , M a c M i l l a n Co., New York, 1925. Aston, R.E., C e l l u l a r C o n t i n u i t y and Development, S c o t t Foresihan and Co., Glenview, 111., 1967.  29. 30.  Robertson, O.H., F i s h e r y B u l l .  10, 137, 1962.  127, F i s h and W i l d l i f e  S e r v i c e , U.S. Dept. I n t . , Washington, 1958. 31.  Henderson, N.E., Can. J . Z o o l . 40_, 631, 1962.  32.  Kodani, M. and K. Kodani, Proc. Nat. Acad. S c i . (U.S.) 56, 1200, 1966.  33.  Lacy, D., Endeavour.26, 101, 1967.  34.  Henderson, N.E., J . F i s h . Res. Bd. Can. 20_, 859 , 1963.  35.  Papkoff, H., D. Gospodarowicz and C H . L i , A r c h . Biochem. Biophys. 120, 434, 1967. Papkoff, H., D. Gospodarowicz, A. C a n d i o t t i and C H . L i ,  36.  A r c h . Biochem. Biophys. I l l , 37.  431, 1965.  Hoar, W.S., Annual Rev. P h y s i o l . 2_7, 51, 1965.  38.. W i t s c h i , E., Mem. Soc. E n d o c r i n o l . 4_, 149, 1955. 39.  B a l l , J.N., Symp. Z o o l . Soc. London 1, 105, 1960.  40.  Otsuka, S., E n d o c r i n o l . Japon. 3_, 272, 1956.  41.  Dodd, J.M., Mem. Soc. E n d o c r i n o l . 7, 17, 1960.  -124  -  42.  Brown, M.E. , The P h y s i o l o g y of F i s h e s _1, Academic New York, p. 447, 1957. .  Press,  43.  Hoar, W.S., i n The P i t u i t a r y Gland, V o l . 1, G.W. H a r r i s and B.T. Donovan, eds., Butterworths, London, 19 64.  44.  Savard, K. and N.R. Mason, A b s t r . E n d o c r i n o l . Soc. 45th Meeting, p. 32, 1963.  45.  Ludwig, D.J., E n d o c r i n o l o g y £ 6 , 453, 1950.  46.  I d l e r , D.R., P.J. Schmidt and I . B i t n e r s , Can. J . Biochem. P h y s i o l . 39, 1653, 1961.  47.  Schmidt, P.J. and D.R. I d l e r , Gen. Comp. E n d o c r i n o l . 2, 204, 1962.  48.  I d l e r , D.R., P.J. Schmidt and J . B i e l y , Can. J . Biochem. P h y s i o l . 3_9, 317, 1961.  49.  I d l e r , D.R., I . B i t n e r s and P.J. Schmidt, Can. J . Biochem. P h y s i o l . 3_9, 1737, 1961.  50.  Emanuel, C F . and I.L. C h a i k o f f , J . B i o l . Chem. 203 , 167, 1953.  51.  Ronald, A.P., and M. Smith, p e r s o n a l  communication.  52.  T a r r , H.L.A., Can. J . Biochem. £ 2 , 51, 1964.  53.  T a r r , H.L.A. , Can. J . Biochem.  54.  T a r r , H.L.A., Can. J . Biochem. _42, 1535, 1964.  55.  T a r r , H.L.A. and J . Roy, Can. J . Biochem.  56.  T a r r , H.L.A. and J . Roy, Can. J . Biochem. 44_, 1453, 1966.  57.  Menon, K.M.J, and M. Smith, p e r s o n a l  58.  McDonald, M.R.,  59. 60.  Smith, M., p e r s o n a l communication. Dixon, C H . and M. Smith, Prog. N u c l e i c A c i d Res. Mol. B i o l . , J.N. Davidson and W.E. Cohn, eds., i n p r e s s , 1968.  61.  B o v e r i , T., V e r h a n d l . Phys.-Med. Ges. Wurz., 35, 1902.  62.  Mirsky, A.E...and H. R i s , Nature 163, 666, 1949.  63.  Stedman, E. and E. Stedman, Nature 166., 780, 1950.  42, 575, 1964.  44, 197, 1966.  communication.  J . Gen. P h y s i o l . Suppl. 4_5, 77, 1962.  - 125 64. "65.  Stedman, E. and E. Stedman, P h i l . Trans. Roy. Ser. B. 235, 565, 1951.  Soc. London,  Huang, R.C. and J . Bonner, Proc. Nat. Acad. S c i . (U.S.) 48, 1216, 1962.  66.  Bonner, J . , M.E. Dahmus, D.M. Fambrough, R.C. Huang, K. Marushige and D.Y.H. Tuan, S c i e n c e , i n p r e s s , 1967.  67.  Bonner, J . , R.C. Huang and R."Gilden, Proc. Nat. Acad. S c i . (U.S.) 50, 893, 1963.  68.  Sonnenberg, B.P. and G. Zubay, Proc. Nat. Acad. S c i . (U.S.) 54, 415, 1965.  69.  B u t l e r , J.A.V.,and A. R.--Chipperf i e l d , Nature  215 , 1188,  1967. 70.  P a u l , J . and R.S.  Gilmour,  J . Mol. B i o l . _16, 242,  1966.  71. 72.  P a u l , J . and R.S. Gilmour, Nature 210, 992, 1966. Fambrough, D.M. and J . Bonner, B i o c h e m i s t r y _5, 2563,  73.  Fambrough, D.M.,  74.  Allfrey, V.G., R. Faulkner and A.E. Mirsky, Proc. Acad.. S c i . (U.S.) 51, 786, 1964.  75.  K l e i n s m i t h , L . J . , V.G. A l l f r e y and A.E. M i r s k y , Proc. Acad. S c i . (U.S.) 55, 1182, 1966.  Nat.  76.  S t e v e l y , W.S.and L.A.  1966.  77.  G u t i e r r i e z , R.,and L.S. H h i l i c a , S c i e n c e 151_  78.  Huang, R.C. and J . Bonner, Proc. Nat. Acad. S C i . (U.S.) 5_4, 960, 1965.  79.  Bonner, J . and J . Widholm, Proc. Nat. Acad. S c i . (U.S.) 57, 1379, 1967.  80.  Huang, R.C. , Fed. Proc. 2_6, 603,  81.  Langan, T.A., Biochim. Biophys. A c t a L i b r a r y , V o l . 10, p. 233, V.V. Koniggsberger and L. 3osch eds., E l s e v i e r , Amsterdam, 1967.  82. j  F r e n s t e r , J.H., i n The C e l l Nucleus-Metabolism and Radios e n s i t i v i t y , T a y l o r and F r a n c i s , London, p. 27, 1956.  83.  Jacob, F. and J . Monod, J . Mol. B i o l .  1966.  F. Fujimura and J . Bonner, i n p r e s s ,  Stocken, Biochem. J . 100, r  1967.  Nat.  20c,  1324,  1967.  1967.  3, 318,  1961.  -  126 -  84.  C l e v e r , U. , Science 146, 794, 1964.  85.  McKusick, V.A. , Quart.  86.  B l o c h , D.P., P r o t o p l a s m a t o l o g i a Vienna, 1966.  87.  F r a s e r , R.C., E x p t l . C e l l Res.- 33, 473, 1964.  88.  Feughelman, M., R. Langridge, W.E. Seeds, A.R. Stokes, H.R. Wilson, P.W. Hooper, M.H.F. W i l k i n s , R.D. B a r c l a y and L.D. Hamilton, Nature 175, 834, 1955.  89.  Zubay, G. and M.H.F. W i l k i n s , J . Mol. B i o l . _4 , 444 , 1962.  90.  Inuoe, S. and H. Sata, i n M o l e c u l a r A r c h i t e c t u r e i n C e l l P h y s i o l o g y , T. Hayashi and A.G. Szent-Gyorgyi, eds., P r e n t i c e H a l l , New York, p. 209, 1966.  91.  Skalka, A., A.W. Fowler and J . Hurwitz, 241, 588, 1966.  J . B i o l . Chem.  92.  B l o c h , D.P. and H.Y.C. Hew, J . Biophys.  Biochem. C y t o l .  Rev. B i o l .  3T7, 69, 1962.  5_, 3d, S p r i n g e r - V e r l a g ,  8, 69, 1960.  93.  Moldave, K. , Annual Rev. Biochem. 3_4, 419, 1965.  94.  Hoagland, M.B., Biochem. Biophys. A c t a 16. i 288, 1955.  95.  C r i c k , F.H.C., Symp. Soc. E x p t l . B i o l . 02, 138, 1958.  96.  Khorana, H.G. , Fed. Proc. 24, 1473, 1965.  97. 98.  Zubay, G., Science 140, 1092, 1963. S i n g e r , M.F. and P. Leder, Annual Rev. Biochem. 35, 195, 1966.  99.  Orczy, Baroness Emmuska, The S c a r l e t Pimpernel, man's Sons, London, 1908.  G.P. Put-  100.  Stedman, E. and E. Stedman, C o l d Spr. Harb. Symp. Quant. B i o l . 12, 224, 1947.  101.  P o l l i s t e r , A.W. and A.E. Mirsky, J . Gen. P h y s i o l . 30, 101, 1946.  102.  Schweet, R., H. Lamfrom and E. A l l e n , Proc. Nat. Acad. S c i . (U.S.) _4_4, 1029, 1958 .  103.  von E h r e n s t e i n , G., B. Weisblum and S. Benzer, Proc. Nat. Acad. S c i . (U.S.) 49, 669, 1963.  - 127 104.  Wagle, S.R., Biochim. Biophys. A c t a 95_, 180, 1965.  -105.  Adiga, P.R., P.M. Rao, R.O. Hussa and T. Winnick, i s t r y 5, 3850, 1966.  106.  N i r e n b e r g , M. and J.H. M a t t h a e i , Proc. Nat. Acad. S c i . (U.S.) 47, 1558, 1961.  107.  N i r e n b e r g , M. and P. Leder, S c i e n c e 14 5,-  108.  Brimacombe, R., J . T u r p i n , M. N i r e n b e r g , P. Leder, M. B e r n f i e l d and T. J a o u n i , Proc. Nat. Acad. S c i . (U.S.) 54, 954, 1965.  109.  Morgan, A.R., R.D. W e l l s and H.G. Khorana, P r o c . Nat. Acad. S c i . (U.S.) 5_6, 1899, 1966.  110.  N i r e n b e r g , M., P. Leder, M. B e r n f i e l d , R. Brimacombe, J . T u r p i n , F. Rottman and C O'Neal, Proc. Nat. Acad. S c i . (U.S.) 5_3, 1161, 1965.  111.  Weisblum, B., F. Gonana, G. von E h r e n s t e i n and S. Benzer, Proc. Nat. Acad. S c i . (U.S.) 53, 328, 1965.  112.  Weisblum, B., J.D. C h e r a y i l , R.M. Bock and D. S o l i , J . Mol. B i o l . _28, 275, 1967.  113.  M a r s h a l l , R.E., C.T. Caskey and M. N i r e n b e r g , Science 155, 820, 1967.  114.  Yanofsky,  115.  Reichmann, M.E., Proc. Nat. Acad. S c i . (U.S.) 5_2, 1009, 1964.  116.  C l a r k , J.M., A.Y. Chang, S. Spiegelman and M.E. Reichmann, Proc. Nat. Acad. S c i . (U.S.) 54, 1193, 1965.  117.  T s u y u k i , H., P.J. Schmidt and M. Smith, J . F i s h . Res. Bd. Can. 21, 635, 1964.  118.  Schmidt, P.J., B.S. M i t c h e l l , M. Smith and H. T s u y u k i , Gen. Comp. E n d o c r i n o l . 5_, 197, 1965.  119.  C u l l i n g , C.F.A., Handbook of H i s t o p a t h o l o g i c a l Butterworths, London, p. 40, 1963.  Techniques,  120.  C u l l i n g , C.F.A., Handbook of H i s t o p a t h o l o g i c a l  Techniques,  Biochem-  1399, 1964.  C , Biochem. Biophys. Res. Commun. _18, 898,  Butterworths, London, p. 36,  1965.  1963.  121.  C r u f t , H.J., Biochem. J . 8*4, 47p, 1962. .  122.  S m i t h i e s , 0., Advances i n P r o t e i n Chemistry 14, 65,  1959.  - 128 123.  J e r g i l , B.G., p e r s o n a l communication.  124.  Bray, G.A., A n a l . Biochem. 1, 279, 1960.  125.  Satake,  K. and J.M. Luck, B u l l . Soc. Chim. B i o l . 4j0, 1743,  1958. 126.  Izumi, Y., A n a l . Biochem. _10, 218, 1965.  127.  Gray, W.R. and B.S. H a r t l e y , Biochem. J . 8j), 59p, 1963.  128.  Black, J.A., unpublished methods, 1965.  129.  Konigsberg, W. and R.J. H i l l , J . B i o l . Chem. 237, 2547, 1962. Robertson, O.H. and S.P. R i n f r e t , E n d o c r i n o l . 6_0, 559,  130.  1957. 131.  G o l d s t e i n , A., B i o s t a t i s t i c s , MacMillan,  13.2. 133.  Raymond, S. and L. Weintraub, Science 130, 711, 1959. J o v i n , T., A. Chrambach and M.A. Naughton, A n a l . Biochem. 9,  New York, 164.  351, 1964.  134.  B l o c h , D.P. and S.D. Brack,  135.  Mach, B., E. Reich and E.L. Tatum, Proc. Nat. Acad. S c i . (U.S.) 5_0, 175, 1963. Yukoida, M., Y. Tsukamoto, Y. S a i t o , T. T s u j i , S. O t a n i and S. O t a n i , Biochem. Biophys. Res. Commun. 19, 204, 1965.  136.  137.  J . C e l l . B i o l . 22, 327,  Uemura, I . , K. Okuda and T. Winnick, B i o c h e m i s t r y 1963.  1964.  2, 719,  138. „ H a l l , J.B., J.W. Sedat, P.R. Adiga, I . Uemura and T. Winnick, J . Mol. B i o l . 12, 162, 1965. 139... Bhagavan, N.V. , P.M. Rao, L.W. P o l l a r d , R.K. Rao, T. Winnick and J.B. H a i l , B i o c h e m i s t r y 5_, 3844, 1966. 140.  Tomino, S., M. Yamada, H. I t o h and K. Kurahashi, i s t r y 6, 2552, 1967.  141.  O t a n i , S., T. Yamanoi, Y. S a i t o and S. O t a n i , Biochem. Biophys.  Res. Commun. 25, 590,  Biochem-  1966.  142.  Humbel, R.E., Proc. Nat. Acad. S c i . (U.S.) 5_3, 853, 1965.  143.  Adiga, P.R., L. Uemura and T. Winnick, B i o c h e m i s t r y _4, 246, 1965.  - 129 144.  K l e t t , R.P., p e r s o n a l  communication.  145.  Block, R.I., D. B o i l i n g , H. Gershon and H.A. Sober, Proc. Soc. E x p t l . B i o l . Med. 70, 494, 1949. '  146.  Goppald-Krekels, A., and H. Lehmann, Z. P h y s i o l . Chem. 313, 147, 1958.  147.  I n g l e s , C.J., J.R. T r e v i t h i c k , M. Smith and G.H. Dixon, Biochem. Biophys. Res. Commun. 26_, 627, 1966, and J.R. T r e v i t h i c k , unpublished r e s u l t s , 1966.  148.  Bennett, L.D., D. Smithers and C.T. Ward, Biochim. Biophys. A c t a 87, 60, 1964.  149.  Goldberg, I.H., Am. J . Med. 39,- 722, 1965.  150.  W a l l e r , C.W., P.W. F r y t h , B.L. Hutchings and J.H. Willams, J . Am. Chem. Soc. 75./ 2025, 1953. Yarmolinsky, M.B. and G.L. de l a Haba, Proc. Nat. Acad. S c i . (U.S.) 4j>, 1721, 1959.  151. 152.  M o r r i s , A . J . , S. F a v e l u k e s , R. A r l i n g h a u s and R. Schweet, Biochem. Biophys. Res. Commun. 1_, 326,  1962.  153.  Nathans,  D., Proc. Nat. Acad. S c i . (U.S.) 51, 585, 1964.  154.  Nathans,  D., Fed. Proc. 23,  155. 156.  E n n i s , H.L., and M. L u b i n , S c i e n c e 146, 1474, 1964. Colombo, B., L. F e l i c e t t i and C. B a g l i o n i , Biochem. Biophys. Res. Commun. 1_8, 389, 1965.  157.  W e t t s t e i n , F.O., H. N o l l and S. Penman, Biochim. Biophys. A c t a 87, 525, 1964.  158.  Nathans,  159.  Wolfe, S.M. and A.S. Weisberger, Proc. Nat. Acad. S c i .  984,  1964.  D., and A. N e i d l e , Nature 197, 1076,  1963.  (U.S.) 5_3, 991, 1965. 160.  T r a u t , R.R. and R.E. Munro, J . Mol. B i o l .  161.  Vazquez,  162.  Wolfe, A.D. and F.E. Hahn, Biochim. Biophys. Acta 95, 146, 1965. Monesi, V., E x p t l . C e l l Res. 36, 638, 1965.  163.  10_, 63,  1964.  D., Biochem. Biophys. Res. Commun. 1_5, 464, 1964.  - 130 164.  Reich, E. and I.H. Goldberg, i n Prog. N u c l . A c i d Res. and Mol. B i o l . , J.N. Davidson and W.E. Cohn eds., Academic P r e s s , New York, V o l . 3, p. 183, 1964.  165.  Lipmann, F., i n Prog, i n N u c l . A c i d Res., J.N. Davidson and W.E. Cohn eds., Academic P r e s s , New York, V o l . 1, p. .135, 1963.  166.  Steward, J.A. and J . Papaconstantinou, Proc. Nat. Acad. S c i . (U.S.) 58, 95, 1967.  167.  S t o c k d a l e , F.E. and Y.J. Topper, Proc. Nat. Acad. S c i . (U.S.) 5_6, 1283, 1966.  168.  I n f a n t e , A.A.  and M. Nemer, Proc. Nat. Acad. S c i . (U.S.)  5_8, 681,  1967.  169.  Gross, P.R.,  J . Expt. Z o o l . 157, 21,  170. 171.  Gross, P.R. and G.H. Cousineau, E x p t l . C e l l Res. 3_3, 386, 1964. S p i r i n , A.S., i n C u r r e n t T o p i c s i n Develop. B i o l . , A. Monroy and A.A. Moscona, Academic P r e s s , New York, V o l . 1, p. 1, 1966.  172.  Schwartz, A.N.  and B.A.  1964.  Zabin, A n a l . Biochem. 1£, 321,  1966. 173.  Porath, J . , Biochim. Biophys. A c t a 39., 193,  174.  G e l o t t e , B., J . Chromatog. 2'  175.  Miranda, F., H. Rochat and S. L i s s i t z k y , J . Chromatog. 1_, 142,  3 3 0  '  1960.  I960.  1962.  176.  Janson, J . C , J . Chromatog.  177.  Ando, T. and F. Sawada, J . Biochem.  178.  Tsuyuki, H. and E. Roberts, J . F i s h . Res. Bd. Can. 20, 101, 1963. Clemens, W.A. , Trans. Roy. Soc. Can., Ser. 3, S e c t . V,  179.  No.  47, p. 1,  2_8, 12,  1967. (Tokyo) £ 8 , 886,  1960.  1953.  180.  Murray K., p e r s o n a l  181.  Markham, R.,  182.  Schroeder, W.A., R.T. Jones, J . Cormick and K. M c C a l l a , A n a l . Chem. 34> 1570, 1962.  private  communication. communication.  - 131 183.  Gomori, C , J . Lab. C l i n . Med. 27_, 955, 1942.  184.  Ames, B.N., i n Methods i n Enzymology, p. 115, 1966.  185.  Lipmann, F . a n d 1958.  186.  Sidbury, J . and V.A. N a j j a r , J . B i o l . Chem. 227, 517, 1957.  187.  Agren, G. and L. Engstrom, A c t a Chem. Scand. 10_, 489, 1956.  188.  Rail,  189.  W o s i l a i t , W.D.,  190.  Krebs, E.G. and E.H. F i s c h e r , Adv. Enzymol. 24, 263,  New York, V o l . 8,  P.A. L e v i n e , J . B i o l . Chem. 231, 277,  T.W., E.W. S u t h e r l a n d , and w'.D. W o s i l a i t , J . B i o l . Chem. 218, 483, 1956. J . B i o l . Chem. 233, 597, 1958.  1962. 191.  Larner, J . and F. Sanger, J . Mol. B i o l .  11, 491, 1965.  192. 193.  Mano, Y. and F. Lipmann, J . B i o l . Chem. 241, 3822, 1966. Lipmann, F., Biochem. Z. 262, 3, 1933.  194.  C a n f i e l d , R.E. and C B . A n f i n s o n , i n The P r o t e i n s , 2nd Edtn., H. Neurath ed., Academic P r e s s , New York, V o l . 1, p. 311, 1963. ,  195.  Hashimoto, C , J . Chem. Soc. Japan, Pure Chem. Sect. 80, 441, 1959.  196.  Allfrey,  V . C , Can. Cancer Conf. 6, 313, 1964.  197.  Marchis-Mouren, G. and F. Lipmann,  Proc. Nat. Acad. S c i .  (U.S.) 53, 1147, 1965. 198.  Langen, T.A. and L.K. Smith, Fed. Proc. 26_, 603, 1967.  199.  J e r g i l , B.C  200. 201.  Singh, V.N., S.S. Dave and T.A. Venkitasubramanian, Lawford, Biochem. CR. J and . 104, H. Schachter, 48c, 1967. J . B i o l . Chem. 241, 5408, 1966.  202.  T r e v i t h i c k , J.R., C J . I n g l e s , and C H . Dixon, Fed. Proc. 26, 603, 1967.  and C H . Dixon, i n p r e p a r a t i o n .  - 132 203.  L i n g , V., J.R. T r e v i t h i c k and G.H. Dixon, i n p r e p a r a t i o n .  .204.  N e e l i n , J.M. and G.C. B u t l e r , Can. J . Biochem. 39_, 485, 1961.  205.  N e e l i n , J.M., P.X. C a l l a h a n , D.C. Lamb and K. Murray, Can. J . Biochem. 42_, 1743, 1964.  "206.  Johns, E.W.,  207.  Reid, B.R. and R.D. C o l e , Proc. Nat. Acad. S c i . (U.S.) 51, 1044, 1964.  208.  A l l f r e y , V.G., J.W. Hopkins, J.H. F r e n s t e r and A.E. Mirsky, Ann. N.Y. Acad. S c i . 8J3, 722, 1960.  209.  A l l f r e y , V.G., R. Meudt, J.W. Hopkins and A.E. M i r s k y , Proc. Nat. Acad. S c i . (U.S.) 4_7, 907, 1961.  210.  Robbins, E. and T.W. Borun, Proc. Nat. Acad. S c i . (U.S.) 57, 409, 1967.  211.  P h i l i p p s , G.R. and J . West, Biochim. Biophys. A c t a 91, 416, 1964.  212.  Busch, H., i n H i s t o n e s , Ciba Found. Study Group No. 24, A.V.S. deReuck and J . Knight eds., L i t t l e , Brown, Boston, p. 88, 1966.  213.  M i l s t e i n , C P . , Nature 215, 1190, 1967.  214.  Brown, J.R. and B.S. H a r t l e y , Biochem. J . 9j8, 59p, 1963.  215.  Ord, M.G. and L.A. Stocken, Biochem. J . 103, 5p, 1967.  216.  Umana, R., S. Updike, J.. R a n d a l l and A.L. Dounce, i n The N u c l e o h i s t o n e s , J . Bonner and P. Ts'o eds., Holden-Day, San F r a n c i s c o , p. 200, 1964.  217.  M i e s l e r , M.H. and T.A. Langen, J . C e l l B i o l . 35./ 91A, 1967.  Physiol.  Biochem. J . 92^, 55, 1964.  

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0093558/manifest

Comment

Related Items