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A pharmacological, chromatographic, and chemical study of the neurohypophysial principles of two elasmobranch… Heinicke, Elizabeth Anne 1972

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A PHARMACOLOGICAL, CHROMATOGRAPHIC, AND CHEMICAL STUDY OF THE NEUROHYPOPHYSIAL PRINCIPLES OF TWO ELASMOBRANCH SPECIES: RAIA RHINA AND SQUALUS ACANTHIAS (PACIFIC VARIETY) by ELIZABETH ANNE HEINICKE B.Sc.(Hon.) , U n i v e r s i t y of Manitoba, 1 Q62 M.Sc., U n i v e r s i t y of Manitoba, 19^5 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of Zoology We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF Affuly, BRITISH 1972 . COLUMBIA In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and s tudy . I f u r t h e r agree t h a t permiss ion f o r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department o r by h i s r e p r e s e n t a t i v e s . It i s understood that copying o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l a i n s h a l l not be a l lowed wi thout my w r i t t e n p e r m i s s i o n . Depa rtment The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada ABSTRACT A wide and conf u s i n g array of neurohypophysial p r i n c i p l e s has been a t t r i b u t e d to the elasmo"branch f i s h ; s e v e r a l of these p r i n c i p l e s are notabl y s i m i l a r , e i t h e r i n t h e i r d i s t i n g u i s h i n g c h a r a c t e r i s t i c s or i n the species i n which they have been r e p o r t e d to occur. Here, an attempt was made to r e s o l v e some of the confusion r e g a r d i n g the exact number and nature of the elasmobranch p r i n c i p l e s , by a study of the p h a r m a c o l o g i c a l , chromatographic, and chem-i c a l c h a r a c t e r i s t i c s of the agents produced by two s p e c i e s : the skate Raia r h i n a and the d o g f i s h Squalus acanthias ( P a c i f i c v a r i e t y ) = Squalus s u c k l e y i . The neurointermediate lobe e x t r a c t s of both species contained a major n e u t r a l o x y t o c i c f r a c t i o n and small amounts of a b a s i c a n t i d i u r e t i c p r i n c i p l e which appeared to be the widespread agent, a r g i n i n e v a s o t o c i n . T h i s i d e n t i f i c a t i o n was based on d i r e c t p harmacological comparison of the p u r i f i e d Squalus p r i n c i p l e a gainst s y n t h e t i c a r g i n i n e v a s o t o c i n . I t was the f i r s t such c h a r a c t e r i z a t i o n of an elasmobranch p r i n c i p l e , and p o s s i b l y of any n a t u r a l l y occur-r i n g o x y t o c i n analogue, against i t s p o s s i b l e s y n t h e t i c e q u i v a l e n t ; e a r l i e r comparisons had been made against the customary o x y t o c i n or v a s o p r e s s i n standards. i i i i i N eurointermediate lobe e x t r a c t s of Raia r h i n a showed an unexpected v a r i a t i o n i n the extent to which t h e i r a c t i v i t y on the i s o l a t e d r a t uterus was p o t e n t i a t e d by the presence of magnesium ions i n the bat h i n g s o l u t i o n (1.8 f o l d to IT.5 f o l d ) . T h i s could not be c o r r e l a t e d with f a c t o r s such as season or l o c a t i o n of the c a t c h ; i t was shown to r e s u l t from anomalies i n the d i s t i n c t i o n which d i f -f e r e n t u t e r i n e p r e p a r a t i o n s made between the unknown peptide and the o x y t o c i n standard. The potency of Raia e x t r a c t also v a r i e d on the m i l k - e j e c t i o n assay, with a corresponding v a r i a t i o n i n the r a t i o of m i l k - e j e c t i o n a c t i v i t y to r a t uterus a c t i v i t y of from k.h to 17-5. Many of the o x y t o c i c p r i n c i p l e s a s c r i b e d to the elasmobranchs have been d i s t i n g u i s h e d by t h e i r degree of magnesium p o t e n t i a t i o n or by the r a t i o of t h e i r m i l k - e j e c t i o n a c t i v i t y to t h e i r o x y t o c i c a c t i v i t y on the r a t u t e r u s . The v a r i a b i l i t y d e s c r i b e d here f o r these two values may i n v a l -i d a t e such d i s t i n c t i o n s ; i n co n j u n c t i o n with other a v a i l -able data, these o b s e r v a t i o n s r a i s e d the p o s s i b i l i t y that the many elasmobranch o x y t o c i n - l i k e p r i n c i p l e s might be reduced to two -- the agent designated as E-^, which d i d not occur i n the two species s t u d i e d here, and g l u m i t o c i n , a p r i n c i p l e of known s t r u c t u r e . L a t e r s t u d i e s on the n e u t r a l f r a c t i o n of Raia r h i n a i n d i c a t e d that the agent was g l u m i t o c i n , but the s t u d i e s on Squalus suggested the i n t r o d u c t i o n of s e v e r a l new p r i n c i p l e s to the elasmobranch spectrum. i v The i d e n t i f i c a t i o n of the Raia p r i n c i p l e as glumi-t o c i n was based on d i r e c t pharmacological comparison of the agent against s y n t h e t i c g l u m i t o c i n , and on chemical a n a l y s i s of the p r i n c i p l e . D i r e c t pharmacological comparison of the Squalus n e u t r a l f r a c t i o n - a g a i n s t s y n t h e t i c g l u m i t o c i n showed c l e a r d i f f e r e n c e s between the two p r i n c i p l e s , n o t a b l y i n t h e i r degree of magnesium p o t e n t i a t i o n and i n t h e i r m i l k -e j e c t i o n a c t i v i t i e s . The Squalus n e u t r a l f r a c t i o n was p u r i -f i e d by g e l f i l t r a t i o n through G-15 Sephadex, by chromatography on Sephadex i o n exchangers, and by chromatography on paper. The a c t i v i t y was separated i n t o two d i s t i n c t f r a c t i o n s on CM Sephadex. These, however, behaved i d e n t i c a l l y throughout numerous comparative procedures; they were subsequently shown to be experimental a r t i f a c t s , and were recombined. The p u r i f i e d n e u t r a l f r a c t i o n showed an a c t i v i t y of 109 IU/mg, the most a c t i v e p r e p a r a t i o n of the Squalus o x y t o c i c agent so f a r obtained. Amino a c i d analyses of the p u r i f i e d m a t e r i a l suggested that i t c o n s i s t s of an equimolar mixture of two p e p t i d e s , n e i t h e r of which has been encountered p r e v i o u s l y i n nature. The work presented here l a y s a b a s i s f o r a b e t t e r understanding of the elasmobranch neurohypophysial p r i n c i p l e s , but the d e t a i l s of the d i s t r i b u t i o n and s t r u c t u r e of these peptides must await yet more extensive s t u d i e s . TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS v LIST OF TABLES . x i i i LIST OF FIGURES xv ACKNOWLEDGEMENT x v i i Chapter 1 - INTRODUCTION 1 I. THE COMPARATIVE BACKGOUND TO THE STUDY OF THE ELASMOBRANCH NEUROHYPOPHYSIAL PRINCIPLES 1 A. The V e r t e b r a t e Hypothalamo-Neurohypophysial Sy st em. . 1 1. Anatomical and H i s t o l o g i c a l C o n s i d e r a t i o n s . 1 2. The Nature of the Neurohypophysial P r i n c i p l e s 5 a. The s t r u c t u r e of the known p r i n c i p l e s . 5 b. The b i o l o g i c a l a c t i v i t i e s of the peptides 7 c. . The p h y l o g e n e t i c d i s t r i b u t i o n of the pept ides 8 B. The Elasmobranch Hypothalamo-Neurohypophysial System i 12 1. Anatomical and H i s t o l o g i c a l C o n s i d e r a t i o n s . 12 2. The Nature of the Elasmobranch Neurohypophysial P r i n c i p l e s 20 a. Evidence f o r neurohypophysial a c t i v i t y i n the elasmobranch p i t u i t a r y 20 v v i Page b. Evidence sup p o r t i n g the e x i s t e n c e of an elasmobranch a n t i d i u r e t i c p r i n c i p l e 25 c. Evidence sup p o r t i n g the e x i s t e n c e of s e v e r a l elasmobranch o x y t o c i c p r i n c i p l e s 26 i . D i f f e r e n c e s between the o x y t o c i c p r i n c i p l e s , based on r e l a t i v e m i l k -e j e c t i o n a c t i v i t i e s 27 i i . D i f f e r e n c e s between the o x y t o c i c p r i n c i p l e s , based on magnesium p o t e n t i a t i o n s 28 i i i . D i f f e r e n c e s between the o x y t o c i c p r i n c i p l e s , based on chromatog-r a p h i c behavior 29 . (1) The r e s o l u t i o n of two o x y t o c i c p r i n c i p l e s w i t h i n a s i n g l e s p e c i e s 29 (a) Species i n which E-j_ and E2 have d i f f e r e n t magnesium p o t e n t i a t i o n s 30 . (b) Species i n which E-^  and E2 have s i m i l a r magnesium p o t e n t i a t i o n s 31 (c) Species i n which n e i t h e r E j nor Eg i s p o t e n t i a t e d by magnesium ions 32 (2) The r e s o l u t i o n of a s i n g l e o x y t o c i c f r a c t i o n w i t h i n each species 32 I I . THE STATEMENT OF THE PROBLEM 38 Chapter 2 - MATERIALS AND METHODS ko . A. C o l l e c t i o n and Storage of the Glands 1+ 0 B. E x t r a c t i o n of the T i s s u e s and Storage of the E x t r a c t h2 C. Methods of B i o l o g i c a l Assay h2 v i i Page 1. Rat Uterus Assay U3 2. Rabbit M i l k - E j e c t i o n Assay kh 3. Frog Bladder Assay H. A n t i d i u r e t i c Assay . k'J 5. A c t i v i t y R a t i o s , Used f o r the Comparison of D i f f e r e n t Assay Methods k9 D. N o n - B i o l o g i c a l Measurements 51 I. Measurement of P r o t e i n or Peptide Content. . 51 2. Measurement of C o n d u c t i v i t y . 53 3. I n a c t i v a t i o n with Sodium T h i o g l y c o l l a t e . . . 53 k. Paper Chromatography 53 5. I s o e l e c t r i c Focusing • 55 E. P u r i f i c a t i o n Procedures 56 1. Gel F i l t r a t i o n .• 56 2. F i l t r a t i o n Through Ion R e t a r d a t i o n Resin . . 58 3. F l a s h Evaporation 59 k. Ion Exchange Chromatography 59 a. D i e t h y l a m i n o e t h y l (DEAE) Sephadex. . . . 60 b. Carboxymethyl (CM) Sephadex. . . . . . . 6 l c. S u l f o e t h y l (SE) Sephadex 65 5. L y o p h i l y z a t i o n (Freeze Drying) 66 6. H y d r o l y s i s and Amino A c i d A n a l y s i s 67 Chapters 3 § RESULTS 69 I. STUDIES OF THE NEUROHYPOPHYSIAL PRINCIPLES OF THE SKATE, RAIA RHINA 69 A. I n t r o d u c t i o n . . . 69 v i i i Page B. Pharmacological and Chromatographic Studies of the Neurointermediate Lobe E x t r a c t s 70 1. B i o l o g i c a l A c t i v i t i e s and A c t i v i t y Ratios of the Crude E x t r a c t . . . 70 2. Studies of the Magnesium P o t e n t i a t i o n of the E x t r a c t s 71 a. The range of magnesium p o t e n t i a t i o n s i n twelve crude e x t r a c t s 71 b. Paper chromatography of e x t r a c t s with d i f f e r e n t magnesium p o t e n t i a t i o n value s 7I+ c. J o i n t assays of e x t r a c t s with d i f f e r e n t .magnesium p o t e n t i a t i o n values 78 d. The i n f l u e n c e of the estrous c y c l e on magnesium p o t e n t i a t i o n values 8 l 3. Comparison of Crude E x t r a c t with S y n t h e t i c G l u m i t o c i n 82 a. Assays of crude e x t r a c t against g l u m i t o c i n 82 b. Assays of crude e x t r a c t s and g l u m i t o c i n against o x y t o c i n 87 C. The P u r i f i c a t i o n of the Extract...... . . . . . . 89 1. E x t r a c t i o n of the,Glands, and P u r i f i c a t i o n of the E x t r a c t by Gel F i l t r a t i o n 89 2. Passage of the E x t r a c t through Ion R e t a r d a t i o n Resin and DEAE Sephadex 91 3. Chromatography of the P r i n c i p l e s on CM Sephadex 92 a. The passage through the column 92 b. The b a s i c f r a c t i o n 95 c. • Rechromatography of the combined n e u t r a l f r a c t i o n on CM Sephadex 96 i.x Page k. Amino A c i d A n a l y s i s o f the O x y t o c i c P r i n c i p l e 98 D. Summary of the S t u d i e s on the N e u r o h y p o p h y s i a l P r i n c i p l e s of R a i a r h i n a . . . . . 102 I I . STUDIES OF THE NEUROHYPOPHYSIAL PRINCIPLES OF THE DOGFISH, SQUALUS ACANTHIAS , (PACIFIC VARIETY). . . 10*1 A. I n t r o d u c t i o n 10k B. P h a r m a c o l o g i c a l and Chromatographic S t u d i e s o f the N e u r o i n t e r m e d i a t e Lobe E x t r a c t s . . . . . . 105 1. B i o l o g i c a l A c t i v i t i e s and A c t i v i t y R a t i o s of the Crude E x t r a c t s 105 2. S t u d i e s of A c e t o n e - E x t r a c t a b l e B i o l o g i c a l A c t i v i t i e s 108 C. The P u r i f i c a t i o n of the E x t r a c t 113 1. E x t r a c t i o n of the Glands I l 6 2. P r e l i m i n a r y P u r i f i c a t i o n on Sephadex G-15- . • l l 6 a. The e x t e n t of p u r i f i c a t i o n . . . 116 b. The minor o x y t o c i c peak 125 c. Paper chromatography of the Sephadex G-15 e l u a t e 125 3. Passage of the E x t r a c t t h r o u g h Ion R e t a r d a t i o n R e s i n . • . 129 k. Passage of the E x t r a c t through DEAE Sephadex . . 129 5. Chromatography on CM Sephadex 130 a. The passage t h r o u g h the column 130 b. The c h e m i c a l a n a l y s i s of the two n e u t r a l peaks 131 c. The p h a r m a c o l o g i c a l a n a l y s i s of the b a s i c peak 135 X Page 6. P u r i f i c a t i o n of the Two N e u t r a l F r a c t i o n s on SE Sephadex 137 7. Comparative Studies of the Two N e u t r a l F r a c t i o n s lkl a. The comparison of the pharmacological a c t i v i t i e s of the n e u t r a l peaks, by assay a-g-adm-stt s y n t h e t i c o x y t o c i n (Syntocinon) ihk b. The comparison of the p h a r m a c o l o g i c a l a c t i v i t i e s of the n e u t r a l peaks., by assay of one f r a c t i o n against the other ihk c. Paper chromatography of the two f r a c t i o n s lk6 d. I s o e l e c t r i c f o c u s i n g of the two f r a c t i o n s 1U8 e. The comparison of the behavior of the two f r a c t i o n s on SE Sephadex. . . . 1U8 f . The ..comparison of the amino a c i d composition of the two f r a c t i o n s 151 g. Rechromatography of f r a c t i o n 1 on CM Sephadex 151 8. The Nature of the S i n g l e N e u t r a l F r a c t i o n . . 155 a. Pharmacological s t u d i e s of the n e u t r a l f r a c t i o n • 155 b. Amino a c i d analyses of the n e u t r a l f r a c t i o n 158 i . The a n a l y s i s of " f r a c t i o n 2" a f t e r p u r i f i c a t i o n on SE Sephadex . . . . . 158 i i . The a n a l y s i s of the combined f r a c t i o n s 159 i i i . The a n a l y s i s of the combined f r a c t i o n s a f t e r paper chromatography 162 x i . Page i v . An i n t e r p r e t a t i o n of the analyses of the n e u t r a l f r a c t i o n l6h D. Summary of the Studies on the Neurohypophysial P r i n c i p l e s of Squalus acanthias 165 Chapter k - DISCUSSION 168 I. STUDIES OF THE NEUROHYPOPHYSIAL PRINCIPLES OF THE SKATE, RAIA RHINA. . 168 1. The V a r i a b i l i t y i n the Apparent B i o l o g i c a l Potency of Crude E x t r a c t s and of G l u m i t o c i n 168 2. The I d e n t i t y of the Neurohypophysial P r i n c i p l e s of Rai a r h i n a • 176 I I . STUDIES OF THE NEUROHYPOPHYSIAL PRINCIPLES OF THE DOGFISH, SQUALUS AC ANTHI AS (PACIFIC VARIETY). .. .: . .. . 178 J 1. The I d e n t i t y of the Basic P r i n c i p l e of Squalus ac a n t h i as • 178 2. C o n s i d e r a t i o n s o o f the I d e n t i t y of the Ne u t r a l P r i n c i p l e of Squalus acanthias . . . . 179 a. Behavior of the n e u t r a l f r a c t i o n on CM Sephadex 179 b. Chromatographic and pharmacological c h a r a c t e r i s t i c s of the n e u t r a l p r i n c i p l e 180 c. Amino a c i d analyses of the n e u t r a l f r a c t i o n 185 d. T h e o r e t i c a l c o n s i d e r a t i o n s and s p e c u l a t i o n s of p o s s i b l e s t r u c t u r e . . . . 190 Chapter 5 - SUMMARY 2 0 0 BIBLIOGRAPHY • 207 APPENDICES I. Source of the Neurointermediate Glands used i n t h i s Study 220 x i i Page I I . Standards used i n B i o l o g i c a l Assays 222 I I I . S o l u t i o n s used i n B i o l o g i c a l Assays. . . . . 223 IV. S o l u t i o n s used i n N o n - B i o l o g i c a l Measurements . . . . . . . 226 BIOGRAPHICAL INFORMATION LIST OF TABLES Table Page I Amino A c i d Sequences of the Known Neuro-h y p o p h y s i a l P r i n c i p l e s g I I The P h y l o g e n e t i c D i s t r i b u t i o n of the Known N e u r o h y p o p h y s i a l P e p t i d e s 3_3 I I I D i s t i n g u i s h i n g C h a r a c t e r i s t i c s of the Elasmobranch O x y t o c i c P r i n c i p l e s 33 IV O x y t o c i c A c t i v i t i e s , w i t h and w i t h o u t Magnesium, of Crude N e u r o i n t e r m e d i a t e Lobe E x t r a c t s of R a i a r h i n a ; and t h e i r Magnesium P o t e n t i a t i o n s 73 V Magnesium P o t e n t i a t i o n s o f P a i r s of R a i a r.hi na N e u r o i n t e r m e d i a t e Lobe E x t r a c t s d u r i n g Independent and J o i n t Rat Ut e r u s Assays . . . . 79 VI A c t i v i t i e s and A c t i v i t y R a t i o s of Crude Ra i a r h i n a N e u r o i n t e r m e d i a t e Lobe E x t r a c t , Assayed A g a i n s t S y n t h e t i c G l u m i t o c i n 83 V I I A Comparison of the A c t i v i t y R a t i o s Found f o r R a i a r h i n a N e u r o i n t e r m e d i a t e . Lobe E x t r a c t when Assayed a g a i n s t S y n t h e t i c G l u m i t o c i n , t o the T h e o r e t i c a l A c t i v i t y R a t i o s E x p ected from M i x t u r e s of G l u m i t o c i n and S m a l l Amounts of B a s i c P e p t i d e • 85 V I I I A c t i v i t i e s and A c t i v i t y R a t i o s of Rai a r h i n a N e u r o i n t e r m e d i a t e Lobe E x t r a c t and of S y n t h e t i c G l u m i t o c i n , Assayed A g a i n s t S y n t h e t i c O x y t o c i n ( S y n t o c i n o n ) 88 IX Amino A c i d A n a l y s e s o f the P u r i f i e d N e u t r a l P r i n c i p l e from R a i a r h i n a 99 X The A c t i v i t i e s and A c t i v i t y R a t i o s of Crude N e u r o i n t e r m e d i a t e Lobe E x t r a c t s of Squalus a c a n t h i a s 106 xi'xi'' x i v Table Page XI The Oxytocic A c t i v i t y E x t r a c t e d from Two Samples of Squalus acanthi as N e u r o i n t e r -mediate Lobes by the Acetone Used f o r C o l l e c t i o n . 110 XII The Magnesium P o t e n t i a t i o n and Chroma-to g r a p h i c Behavior of a Crude E x t r a c t of Squalus ac anthi as Neurointermediate Lobes, and of the A c t i v i t y Recovered from the Acetone i n which the Glands were C o l l e c t e d . . . 112 XIII The F i l t r a t i o n of Squalus acanthi as and Rai a r h i n a Neurointermediate Lobe E x t r a c t Through Sephadex G-15 12U XIV The Chromatography of Squalus ac a n t h i as and Rai a r h i n a Neurohypophysial P r i n c i p l e s on CM Sephadex, F o l l o w i n g P u r i f i c a t i o n on Sephadex G-15 and DEAE Sephadex 13h XV Amino A c i d Analyses of Squalus acanthias N e u t r a l F r a c t i o n 1 and N e u t r a l F r a c t i o n 2 at Two Stages of P u r i f i c a t i o n . . • 136 XVI The A c t i v i t i e s and A c t i v i t y R a t i o s of the Basic Neurohypophysial P r i n c i p l e from Squalus ac a n t h i as 138 XVII The A c t i v i t i e s and A c t i v i t y R a t i o s of the Two N e u t r a l F r a c t i o n s from Squalus a c a n t h i a s . . 1^5 XVIII The A c t i v i t i e s and A c t i v i t y R a t i o s of Squalus acanthias N e u t r a l F r a c t i o n 2 a, Assayed a g a i n s t N e u t r a l F r a c t i o n l a l U T XIX The A c t i v i t i e s and A c t i v i t y R a t i o s of the O x y t o c i n - l i k e F r a c t i o n 3 from Squalus  ac anthi as, Assayed a g a i n s t S y n t h e t i c Glumitocin 157 XX Amino A c i d Analyses of the P u r i f i e d N e u t r a l F r a c t i o n from Squalus acanthias l60 XXI Amino A c i d A n a l y s i s of the Unhydrolyzed, P u r i f i e d N e u t r a l F r a c t i o n 3 from Squalus ac a n t h i as 161 XXII Amino A c i d A n a l y s i s of the H y d r o c h l o r i c A c i d Used f o r H y d r o l y s e s a 163 LIST OF FIGURES F i g u r e Page 1 The c a t , schemat ic l o n g i t u d i n a l s e c t i o n of the p i t u i t a r y g land 1+ 2 Squalus a c a n t h i a s , median d iagrammatic s e c t i o n o f the p i t u i t a r y g l a n d 15 3 Descending paper chromatograms of t h r e e crude n e u r o i n t e r m e d i a t e lobe e x t r a c t s of R a i a r h i n a , on Whatman 3MM p a p e r , i n n - b u t a n o l : a c e t i c a c i d : w a t e r = k:l:5, at room temperature 76 Ua Chromatogram of the p a r t i a l l y p u r i f i e d n e u r o i n t e r m e d i a t e lobe e x t r a c t of R a i a r h i n a on a 35 cm x 0.9 cm column of CM Sephadex. . . . 9I+ Ub Chromatogram of combined f r a c t i o n 1 and f r a c t i o n 2 of R a i a r h i n a ( p u r i f i e d on Sephadex G-15 and on CM Sephadex - see F i g . ha, above) on a second 35 cm x 0.9 cm column of CM Sephadex gk 5 Descend ing paper chromatograms of Squalus  a c a h t h i a s n e u r o i n t e r m e d i a t e lobe e x t r a c t , at v a r i o u s s tages of p u r i f i c a t i o n , on Whatman 3MM p a p e r , i n n - b u t a n o l : a c e t i c a c i d r w a t e r = U : l:5, at room temperature 115 6a G e n e r a l o u t l i n e of the p rocedures f o l l o w e d d u r i n g the p u r i f i c a t i o n of the a c t i v e p r i n c i p l e s from the n e u r o i n t e r m e d i a t e lobe (NIL) of Squalus a c a n t h i a s ( P a c i f i c v a r i e t y ) . . 118 6b D e t a i l e d o u t l i n e of the p rocedures f o l l o w e d d u r i n g the p u r i f i c a t i o n of the a c t i v e p r i n c i p l e s from the n e u r o i n t e r m e d i a t e lobe (NIL) of Squalus a c a n t h i a s ( P a c i f i c v a r i e t y ) . . 120 xv x v i F igure Page 7 Chromatogram of 10 ml of crude Squalus  acanthias neurointermediate lobe e x t r a c t on a 90 cm x 2.h cm column of Sephadex G-15. . . 123 8 Descending paper chromatogram of Squalus  acanthias neurointermediate lobe e x t r a c t ( P r e p a r a t i o n 2, a f t e r p a r t i a l p u r i f i c a t i o n on Sephadex G-15) on Whatman 3MM paper, i n n - b u t a n o l : a c e t i c acid:water = U:l:5, at room temperature 128 9 Chromatogram of p a r t i a l l y p u r i f i e d neuro-i n t e r m e d i a t e lobe e x t r a c t of Squalus ac a n t h i as ( P r e p a r a t i o n 3; Sephadex G-15 — p u r i f i e d ) on a 35 cm x 0.9 cm column of CM Sephadex 133 10a Chromatogram of Squalus a c a n t h i as n e u t r a l f r a c t i o n l a ( f r a c t i o n 1 from P r e p a r a t i o n s 2 and 3, combined; CM Sephadex-purified) on a 35 cm x 0.9 cm column of SE Sephadex- 1U3 10b Chromatogram of Squalus ac a n t h i as n e u t r a l f r a c t i o n 2a ( f r a c t i o n 2 from P r e p a r a t i o n s 2 and 3, combined; CM Sephadex-purified) on a 35 cm x 0.9 cm column of SE Sephadex 11+3 11 I s o e l e c t r i c f o c u s i n g of a mixture of Squalus  ac a n t h i as n e u t r a l f r a c t i o n 1 and n e u t r a l f r a c t i o n 2 ( P r e p a r a t i o n 3, Sephadex G-15-and CM Sephadex-purified) i n a pH range of 7-9 at 800 v o l t s f o r 25 hours 150 12a Chromatogram of p a r t i a l l y p u r i f i e d neuro-in t e r m e d i a t e lobe e x t r a c t of Squalus  acanth i a s ( P r e p a r a t i o n k; Sephadex G-15-p u r i f i e d ) on a 35 cm x 0.9 cm column of CM Sephadex 15U 12b Chromatogram of Squalus ac anthias f r a c t i o n 1 ( P r e p a r a t i o n k; p u r i f i e d on Sephadex G-15 and on CM Sephadex - see F i g . 12a, above -and a l s o on SE Sephadex) on a second 35 cm x 0.9 cm column of CM Sephadex I5U ACKNOWLEDGEMENTS The author wishes to express her s i n c e r e g r a t i t u d e to Dr. A. M. Perks of the Zoology Department f o r h i s super-v i s i o n o f , and encouragement d u r i n g , t h i s i n v e s t i g a t i o n . She would l i k e to thank Mr. V. Swiatkiewicz f o r h i s help i n c o l l e c t i n g the glands. She wishes to thank Dr. G. Somero and Mr. H. B e h r i s c h of the Zoology Department f o r c a r r y i n g out the i s o e l e c t r i c d e t e r m i n a t i o n s . She i s g r a t e f u l to Dr. G. Dixon of the Bioc h e m i s t r y Department and to Dr. 0 . Parkes of the Pharmacology Department f o r the amino a c i d analyses which were performed i n t h e i r l a b o r a t o r i e s . She i s al s o indebted to the N a t i o n a l Research C o u n c i l f o r f i n a n c i a l a s s i s t a n c e i n the form of an N. R. C. Studentship f o r the years 1965-68, and to the H. R. MacMillan f a m i l y f o r an H. R. MacMillan Family F e l l o w s h i p f o r the year 1968-69. x v i i Chapter 1 INTRODUCTION I. THE COMPARATIVE BACKGROUND TO THE STUDY OF THE ELASMOBRANCH NEUROHYPOPHYSIAL PRINCIPLES This study deals with the a c t i v e p r i n c i p l e s produced by the hypothalamo-neurohypophysial system of c e r t a i n elasmobranch s p e c i e s . Although the elasmobranch system i s b a s i c a l l y s i m i l a r to that found i n other v e r t e b r a t e s , there are some important d i f f e r e n c e s . Therefore the general v e r t e b r a t e system w i l l be o u t l i n e d before a more d e t a i l e d d e s c r i p t i o n of the elasmobranch system i s presented. A. The Ve r t e b r a t e Hypothalamo-Neurohypophysial System 1. Anatomical and H i s t o l o g i c a l C o n s i d e r a t i o n s The v e r t e b r a t e hypothalamo-neurohypophysial system c o n s i s t s of aggregations of s e c r e t o r y nerve c e l l s i n the hypothalamic r e g i o n of the b r a i n , of nerve t r a c t s formed by the axons of these n e r v e ' c e l l s , and of t h e i r t e r m i n a t i o n s i n the p i t u i t a r y gland - a r e g i o n known as the neurohypophysis (Bargmann and S c h a r r e r , 1951; Sl o p e r , 1966). The hypothalamic n u c l e i which form a pa r t of t h i s system c o n s i s t of the p a i r e d supraoptic and p a r a v e n t r i c u l a r n u c l e i found i n r e p t i l e s , b i r d s , and mammals, and of the 1 2 homologous p r e o p t i c n u c l e i of the cyclostomes, elasmobranchs, t e l e o s t s , and amphibians (Bargmann and Sc h a r r e r , 1951; Sloper , 1966). Other nerve c e l l s , such as those of the l a t e r a l t u b e r a l n u c l e i , may a l s o be i n v o l v e d i n the system ( C h r i s t , 1966; Sl o p e r , 1966). The axons of the s e c r e t o r y nerve c e l l s form the hypothalamo-hypophysial nerve t r a c t s , which pass i n t o the s p e c i a l i z e d r e g i o n of the i n f u n d i b u l a r f l o o r which forms the neurohypophysis (Bargmann and Sc h a r r e r , 1951; C h r i s t , 1966; Slo p e r , 1966). In dipnoan f i s h e s , amphibians, r e p t i l e s , b i r d s , and mammals the r o s t r a l p o r t i o n of the neurohypophysis contains a s p e c i a l i z e d r e g i o n , the median eminence, from which small p o r t a l blood v e s s e l s pass to the adenohypophysis ( S l o p e r , 1966). Caudal to the median eminence the neurohypophysis t h i c k e n s to form the i n f u n d i b u l a r process ( F i g . l ) . The i n t e r v e n i n g por-t i o n of the neurohypophysis, which i s d i f f i c u l t to d i s t i n g u i s h i n many s p e c i e s , i s the i n f u n d i b u l a r stem ( S l o p e r , 1966). The part s of the neurohypophysis which do not i n c l u d e the median eminence are known as the pars nervosa (Gorbman and Bern, 1962), and w i l l be r e f e r r e d to as such i n t h i s t h e s i s . The nerves of the hypothalamo-hypophysial t r a c t s terminate i n the median eminence and i n f u n d i b u l a r process (reviewed i n C h r i s t , 1966; Green, 1966; Sloper, 1966), and i t i s g e n e r a l l y accepted that the s e c r e t i o n produced by the neu r o s e c r e t o r y c e l l s i s r e l e a s e d from these nerve endings ( S l o p e r , 1966), probably i n t o the blood v e s s e l s . 3 F i g u r e 1. The c a t , schematic l o n g i t u d i n a l s e c t i o n of the p i t u i t a r y gland. From R u s s e l l ( l Q 6 5 ; F i g . l ) . a: o p t i c chiasma, b: t h i r d v e n t r i c l e , c: s u p r a o p t i c n u c l e u s , d: p a r a v e n t r i c u l a r n u c l e u s , e: median eminence, f : pars nervosa (e, f : neurohypophysis), g: pars i n t e r m e d i a , h: pars d i s t a l i s , i : pars t u b e r a l i s (g, h, i : adenohypophysis). 5 The study p r e s e n t e d here i s concerned w i t h the nat u r e of the s e c r e t i o n which i s r e l e a s e d from the i n f u n d i -b u l a r p r o c e s s (pars n e r v o s a ) . I t can be t r a c e d throughout the n e u r o s e c r e t o r y system as a s t a i n a b l e c o l l o i d when viewed by l i g h t m i c r o s c o p y , or as s m a l l e r n e u r o s e c r e t o r y g r a n u l e s when seen by e l e c t r o n m i c r o s c o p y ; i t has been a s s o c i a t e d w i t h the b i o l o g i c a l a c t i v i t y ( v a s o p r e s s o r , a n t i d i u r e t i c , and o x y t o c i c ) which has been found i n a l l p a r t s of the system (Bargmann, i 9 6 0 ) . In p a r t , at l e a s t , i t c o n s i s t s of the a c t i v e n e u r o h y p o p h y s i a l p r i n c i p l e s i n a s s o c i a t i o n w i t h a c a r r i e r p r o t e i n , " n e u r o p h y s i n " (Chauvet et a^l, , 1960a). I t i s s t o r e d i n nerve endings i n the i n f u n d i b u l a r p r o c e s s , from which i t i s r e l e a s e d i n t o the b l o o d stream (Bargmann and S c h a r r e r , 1951). 2. The Nature of the N e u r o h y p o p h y s i a l P r i n c i p l e s a. The s t r u c t u r e of the known p r i n c i p l e s A l l the n e u r o h y p o p h y s i a l p r i n c i p l e s so f a r i s o l a t e d and a n a l y z e d are o c t a p e p t i d e s . T h e i r s t r u c t u r e s are l i s t e d i n Table I . I t can be seen t h a t a l l the p e p t i d e s have the same b a s i c s t r u c t u r e , w i t h d i f f e r e n c e s between t h e i r mole-c u l e s o c c u r r i n g i n amino a c i d p o s i t i o n s 3, ht and 8 (see Sawyer, 1968). However the two v a s o p r e s s i n s and a r g i n i n e v a s o t o c i n are c h e m i c a l l y b a s i c p e p t i d e s w h i l e the o t h e r s are more n e a r l y n e u t r a l . T h i s d i f f e r e n c e i s i m p o r t a n t and u s e f u 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 the d i f f e r e n t p r i n c i p l e s . 6 Table I Amino A c i d Sequences of the Known Neurohypophysial P r i n c i p l e s A r g i n i n e v a s o p r e s s i n ( l , 2 ) a Cys. Tyr. Phe. Glu(NHp). Asp(NH„). Cys. Pro. Arg. Gly(NHg) 1 2 3 k 5 6 7 8 9 Lysine v a s o p r e s s i n (2, 3) Cys. Tyr. Phe. Glu(NH 2). Asp(NH 2). Cys. Pro. Lys. Gly(NHg) A r g i n i n e v a s o t o c i n (k, 5 9 6, 7 S 8, 9) Cys. Tyr. I l e u . Glu(NH„). Asp(NH Q). Cys. Pro. Arg. Gly(NH P) Oxytocin (10, 11, 12) Cys. Tyr. I l e u . G I U ( N H Q ) . Asp(NHp). Cys. Pro. Leu. G l y ( N H 2 ) Mesotocin ( 8 - i l e u oxytocin) (13) Cys. Tyr. I l e u . Glu(NHg). Asp(NH 2). Cys. Pro. I l e u . Gly(NH 2) I s o t o c i n ( U - s e r , 8 - i l e u oxytocin) (9» 1*0 Cys. Tyr. I l e u . Ser .Asp(NH 2). Cys. Pro. I l e u . Gly(NH 2) Glu m i t o c i n (l(-ser , 8-glu(NH 2) oxytocin) (15) Cys. Tyr. I l e u . Ser .Asp(NH 2>. Cys. Pro. Glu(NR" 2). Gly (NH 2) R e f e r e n c e s : 1: Acher and Chauvet (1953), 2: du Vigneaud, R e s s l e r , and T r i p p e t t (1953), 3: Popenoe et_ a l . (1952), h: Katsoyannis (1958), 5: Chauvet et_ a l . (1960b), 6: Acher e t a l . ( i 9 6 0 ) , 7: Rasmussen and 'Craig (1961), 8: H e l l e r and P i c k e r i n g (1961), 9: Wilson and Smith (1969), 10: du Vigneaud, R e s s l e r , Swan, Roberts, Katsoyannis, and Gordon (1953), 11: du Vigneaud, Lawler, and Popenoe (1953), 12: du Vigneaud (1956), 13: Acher et_ a l . (196*0 , lh : Acher et_ a l . (1962), 15: Acher et a l . (1965). 7 b . The b i o l o g i c a l . a c t i v i t i e s o f t h e p e p t i d e s B i o l o g i c a l a c t i v i t i e s c h a r a c t e r i s t i c o f t h e s e p e p -t i d e s h a v e b e e n k n o w n f o r many y e a r s . O l i v e r a n d S c h a f e r (1895) f i r s t o b s e r v e d t h a t t h e i n j e c t i o n o f p i t u i t a r y e x t r a c t s i n t o t h e d o g c a u s e d a r a p i d r i s e i n b l o o d p r e s s u r e . A d e c a d e l a t e r , D a l e (1906) f o u n d t h a t p i t u i t a r y e x t r a c t f r o m t h e ox n o t o n l y p r o d u c e d a r i s e o f b l o o d p r e s s u r e i n t h e p i t h e d e a r l y - p r e g n a n t c a t b u t a l s o c a u s e d i_n_ v i v o c o n t r a c t i o n s o f t h e u t e r u s . He l a t e r (1909) o b s e r v e d a s i m i l a r o x y t o c i c e f f e c t o f ox p o s t e r i o r l o b e (= p a r s n e r v o s a + p a r s i n t e r m e d i a ) e x t r a c t on t h e u t e r u s o f t h e c a t , d o g , g u i n e a p i g , r a t a n d r a b b i t , b o t h i n . v i v o a n d i_n v i t r o . The g a l a c t o b o l i c o r m i l k - e j e c t i o n e f f e c t o f p o s t e r i o r l o b e e x t r a c t s was s e e n b y O t t a n d S c o t t (1910), who f o u n d t h a t i n t r a v e n o u s i n j e c t i o n o f t h e e x t r a c t s i n t o t h e l a c t a t i n g g o a t was f o l l o w e d r a p i d l y b y a g r e a t i n c r e a s e i n t h e r a t e o f m i l k l e t - d o w n . The a v i a n v a s o d e p r e s s o r e f f e c t was o b s e r v e d f i r s t b y P a t o n a n d W a t s o n (1912). T h e y f o u n d t h a t e x t r a c t s i n j e c t e d i n t r a v e n o u s l y i n t o t h e d u c k , a t a d o s e l e v e l w h i c h w o u l d r a i s e t h e b l o o d p r e s s u r e o f t h e r a b b i t a n d c a t , c a u s e d a d e c r e a s e i n t h e b l o o d p r e s s u r e o f t h e b i r d . P o s t e r i o r l o b e e x t r a c t s w e r e a l s o f o u n d t o c a u s e a n t i d i u r e t i c e f f e c t s i n mammals ( v o n d e n V e l d e n , 1913). I n a p r e s u m a b l y a n a l a g o u s m a n n e r , t h e y r e s u l t e d i n w a t e r u p t a k e i n w h o l e a m p h i b i a n s ( H e l l e r , 19 ^ - l a ) , and i n t h e t r a n s p o r t o f w a t e r a c r o s s t h e a m p h i b i a n b l a d d e r ( B e n t l e y , 1958; S a w y e r , i 9 6 0 ) . M o r e r e c e n t l y , a c o n t r a c t i o n 8 of the hen o v i d u c t has "been observed, both in_ v i v o and i n  v i t r o , i n response to neurohypophysial peptides (Munsick et a l . , I960 ) . Although the v a r i o u s neurohypophysial peptides share a number of b i o l o g i c a l a c t i o n s , d i f f e r e n t peptides e x h i b i t c e r t a i n e f f e c t s more s t r o n g l y than o t h e r s . On the b a s i s of t h e i r s t r o n g e s t b i o l o g i c a l a c t i v i t i e s the peptides can be roughly c l a s s i f i e d as e i t h e r o x y t o c i c or vasopressor. This c l a s s i f i c a t i o n corresponds i n a general way to the c l a s s i f i -c a t i o n on the b a s i s of charge. The n e u t r a l peptides ( o x y t o c i n , mesotocin, i s o t o c i n , g l u m i t o c i n ) have strong u t e r o t o n i c ( o x y t o c i c ) and m i l k - e j e c t i o n p o t e n c i e s , and r e l a t i v e l y l i t t l e p r e s s o r or a n t i d i u r e t i c e f f e c t s . On the other hand the b a s i c peptides show high pressor and a n t i d i u r e t i c a c t i v i t i e s . Of these b a s i c vasopressor p r i n c i p l e s , a r g i n i n e and l y s i n e v a s o p r e s s i n have l i t t l e r a t uterus or m i l k - e j e c t i o n potency; however, a r g i n i n e v a s o t o c i n , which contains s t r u c t u r a l elements of both o x y t o c i n and a r g i n i n e v a s o p r e s s i n , has roughly equal o x y t o c i c and p r e s s o r a c t i v i t y (see H e l l e r , 1963; Sawyer, 1965 ; Sawyer et a l . , 1970). c. The p h y l o g e n e t i c d i s t r i b u t i o n of the peptides In g e n e r a l , each c l a s s of v e r t e b r a t e produces one or more of both the b a s i c and the n e u t r a l p e p t i d e s . The c y c l o -stomes, i n which no n e u t r a l peptide has yet been found, are the only exception. The f o l l o w i n g account of the d i s t r i b u -t i o n of the neurohypophysial hormones i s based on c o n s i d e r a t i o n s presented by Sawyer (1968). 9 Oxytocin i s the n e u t r a l peptide produced by a l l mammals so f a r i n v e s t i g a t e d . Most mammals a l s o produce a r g i -nine v a s o p r e s s i n , but a few produce l y s i n e v a s o p r e s s i n i n s t e a d , and some species appear to produce both. Lysine v a s o p r e s s i n appears i n some species of the suborder Suina, which i n c l u d e s p i g s , p e c c a r i e s , and the hippopotamus. Many of these species produce both a r g i n i n e and l y s i n e v a s o p r e s s i n as w e l l as o x y t o c i n . When t h i s i s the case, some i n d i v i d u a l s w i t h i n a given species may produce only one of the v a s o p r e s s i n s , while other i n d i v i d u a l s of the same species appear to sec r e t e both (Ferguson, 1969). Only the domestic hog (Sus) and the bushpig (Potamochoerus porcus ) produce l y s i n e v a s o p r e s s i n e x c l u s i v e l y (Sawyer, 1968; Ferguson and P i c k e r i n g , 1969K Apart from the Suina, l y s i n e v a s o p r e s s i n has a l s o been r e p o r t e d i n the mouse, Mus muscuius, Peru s t r a i n (Stewart, 1968). In a d d i t i o n to o x y t o c i n and v a s o p r e s s i n , some mammalian f o e t u s e s , those of the sheep (Ovis a r i e s ) and of the s e a l ( C a l l o r h i n u s ur s i n u s ) , appear to produce a r g i n i n e v a s o t o c i n i n small amounts ( V i z s o l y i and Perks, 1969). B i r d s produce o x y t o c i n and a r g i n i n e v a s o t o c i n . Chauvet et a l . ( l 9 6 o b ) i d e n t i f i e d a r g i n i n e v a s o p r e s s i n as w e l l as a r g i n i n e v a s o t o c i n i n p i t u i t a r y e x t r a c t s of the chicken ( G a l l u s dome st i cus ) ; but other workers (Munsick et. al.. , I 9 6 0 ; H e l l e r and P i c k e r i n g , 1 9 6 l ; Munsick, I 9 6 U ) could f i n d no pharm a c o l o g i c a l evidence f o r i t s presence i n the same s p e c i e s . Furthermore, Munsick ( 1 9 6 U ) was able to d i s t i n g u i s h the avian 10 p r i n c i p l e s from a r g i n i n e v a s o p r e s s i n by column chromatography. He suggested t h a t the a r g i n i n e v a s o p r e s s i n observed by Chauvet et a l . was i n t r o d u c e d i n t o the chicken e x t r a c t on the mammalian neurophysin that was used i n the i s o l a t i o n of the avian p r i n c i p l e s . A r g i n i n e v a s o t o c i n and mesotocin ( 8 - i s o l e u c i n e o x y t o c i n ) have been found i n r e p t i l e s (Munsick, 1966; Acher et a l . , 1969). In a d d i t i o n , P i c k e r i n g (1967) found that the n e u t r a l f r a c t i o n of the cobra (Naj a naj a) c o n s i s t e d of a mixture of mesotocin and o x y t o c i n . Amphibians also secrete a r g i n i n e v a s o t o c i n and meso-t o c i n (Acher et al^. , i960; 1964; F o l l e t t and H e l l e r , 1964b). However one s p e c i e s , Rana p i p i en s , has been found to produce ox y t o c i n r a t h e r than mesotocin (Munsick, 1966). The a v a i l a b l e evidence i n d i c a t e s that a r g i n i n e v a s o t o c i n , mesotocin and o x y t o c i n a l s o occur among the l u n g -f i s h ( F o l l e t t and H e l l e r , 1964b; P i c k e r i n g and McWatters, 1966; Sawyer, 1969). Among the r a y - f i n n e d f i s h , a r g i n i n e v a s o t o c i n has been i d e n t i f i e d i n the T e l e o s t e i ( H e l l e r and P i c k e r i n g , I96I; Rasmussen and C r a i g , 196l; Acher et. aJL. , 196l; Wilson and Smith, 1969) and there i s evidence that i t i s produced also by the H o l o s t e i and Chondrostei ( F o l l e t t and H e l l e r , 196Ua). The n e u t r a l p r i n c i p l e , i s o t o c i n ( U - s e r i n e , 8 - i s o l e u c i n e oxytocin) has been i d e n t i f i e d i n the T e l e o s t e i (Acher e_i a l .•, 11 1962; Wilson and Smith, 1969) and i t i s thought to occur among the H o l o s t e i ( F o l l e t t and H e l l e r , 1964a). An o x y t o c i n - l i k e f r a c t i o n occurs i n the Chondrostei, but i t s i d e n t i t y has not been determined ( F o l l e t t and H e l l e r , 1964a). The p r i m i t i v e b i c h i r , P o l y p t e r u s s e n e g a l i s , appears to produce the same i s o t o c i n and a r g i n i n e v a s o t o c i n as the higher a c t i n o p t e r y g i a n s (Sawyer, 1969 ) • Among the c a r t i l a g i n o u s f i s h , the n o l o c e p h a l i a n Hydrolagu s c o l l e i ( r a t f i s h ) has been found to produce two p r i n c i p l e s which resemble a r g i n i n e v a s o t o c i n and o x y t o c i n i n t h e i r chromatographic and pharmacological c h a r a c t e r i s t i c s , and i n t h e i r amino a c i d compositions (Sawyer, 1965; Sawyer et a l . , 1967; P i c k e r i n g and H e l l e r , 1969). However there i s a l s o some chromatographic evidence that the o x y t o c i c f r a c t i o n of t h i s s p e c i e s and of two other h o l o c e p h a l i a n s , Chimaera  monstrosa ( r a b b i t f i s h ) , and Callorhynchus sp., c o n s i s t s of two d i s t i n c t o x y t o c i c p r i n c i p l e s ( H e l l e r and Roy, 1965b; 1967; Roy, 1969). Neither has yet been i d e n t i f i e d . The number and nature of the p r i n c i p l e s produced by the elasmo-branchs i s u n c e r t a i n . These w i l l be d i s c u s s e d i n a l a t e r s e c t i o n . A r g i n i n e v a s o t o c i n has been found i n the cyclostomes, but so f a r there i s no evidence f o r the presence of a n e u t r a l p r i n c i p l e (Sawyer, 1968; Rurak and Perks, unpublished o b s e r v a t i o n s ) . 12-Table IT summarizes the known p h y l o g e n e t i c d i s t r i b u t i o n of the neurohypophysial p e p t i d e s . B. The Elasmobranch Hypothalamo-Neurohypophysial System 1. Anatomical and H i s t o l o g i c a l C o n s i d e r a t i o n s The elasmobranch p i t u i t a r y i s notably d i f f e r e n t from the -well-known mammalian p a t t e r n , and so i t w i l l be d e s c r i b e d i n d e t a i l . I t i s shown d i a g r a m a t i c a l l y i n F i g . 2. The adenohypophysis c o n s i s t s of three l o b e s ; the int e r m e d i a t e l o b e , the a n t e r i o r l o b e , and the v e n t r a l lobe (Meurling, 1967a). The in t e r m e d i a t e l o b e , or pars i n t e r -media, at the p o s t e r i o r end of the adenohypophysis , c o n s i s t s of i r r e g u l a r e p i t h e l i a l c e l l columns separated by a dense system of blood s i n u s e s . The a n t e r i o r lobe continues r o s t r a l from the i n t e r m e d i a t e lobe. The v e n t r a l lobe i s i s o l a t e d i n the base of the cranium, and may or may not r e t a i n a connection with the a n t e r i o r lobe. In elasmobranchs, the neurohypophysis i s not r e a d i l y apparent, and f o r some time i t s presence was doubted. H e r r i n g (1908a; 1911; 1913) could f i n d no d i f f e r e n t i a t i o n of the p i t u i t a r y of the skate, Raia b a t i s , i n t o adenohypophysis and neurohypophysis. He found that during the development of the gland there i s no e v a g i n a t i o n of the infundibulum to form a pars nervosa as occurs i n higher forms. However he d i d n o t i c e that the lamina of nervous t i s s u e which l i e s i n the i n f u n d i b u l a r f l o o r above the p i t u i t a r y "passes i n t o " the Tab le II The P h y l o g e n e t i c D i s t r i b u t i o n of the Known N e u r o h y p o p h y s i a l - P e p t i d e s V e r t e b r a t e type Neurohypophys ia l p e p t i d e A r g i n i n e Lys ine . A r g i n i n e Oxy toc in Metp - I s o - Glumi-v a s o p r e s s i n v a s o p r e s s i n v a s o t o c i n t o c i n t o c i n t o c i n Mammals B i r d s R e p t i l e s Amphibians Bony f i s h l u n g f i s h r a y - f i n n e d f i s h b i c h i r + + (?) + + + + + + + + + + + + + C a r t i l a g i n o u s f i s h ho lo c e p h a l i an s e lasmobranchs + + Cyclostomes Ik F i g u r e 2. Squalus .acanthias , median diagrammatic s e c t i o n of the p i t u i t a r y gland. 'From Meurling (1962 ;-F i g . 1 and 1967a; F i g . 2). a: o p t i c chiasma, b: t h i r d ventricle,-' c: median eminence-,-d: pars nervosa ( c , d:- neurohypophysis), e: pars i n t e r m e d i a (d, e: neurointermediate l o b e ) , f : a n t e r i o r l o b e , g: v e n t r a l lobe (e, f , g: adenohypophysis) , h: saccus. vascu-l o s u s , i : cranium. 15 16 t i s s u e o f t h e p i t u i t a r y . O t h e r w o r k e r s a l s o o b s e r v e d n e r v o u s t i s s u e i n t h e e l a s m o b r a n c h a d e n o h y p o p h y s i s . S t e r z i (1909) and B a u m g a r t n e r (l915)» b o t h w o r k i n g on t h e d o g f i s h , S q u a l u s ' a c a n t h i a s , f o u n d numerous l a r g e s o l i d b u n d l e s o f n e r v e f i b e r s p a s s i n g i n t o t h e t i s s u e o f t h e p a r s i n t e r m e d i a f r o m t h e f l o o r o f t h e b r a i n a b o v e . The b u n d l e s were s e p a r a t e d f r o m t h e .pars i n t e r m e d i a by a c o n n e c t i v e t i s s u e membrane. Then i n 1925 Hogben and de B e e r o b s e r v e d a s m a l l b u t d e f i n i t e mass o f f i b e r s l e a v i n g t h e i n f u n d i b u l a r f l o o r i n t h e s k a t e ( s p ? ) . I n t h i s c a s e t h e f i b e r s r a m i f i e d among t h e c e l l s o f t h e p a r s i n t e r m e d i a . Hogben and de B e e r s u g g e s t e d t h a t t h i s d i f f u s e s t r u c t u r e m i g h t be a t y p e o f p a r s n e r v o s a . The e x i s t e n c e o f t h e e l a s m o b r a n c h p a r s n e r v o s a was f i r m l y e s t a b l i s h e d i n 1952 by S c h a r r e r , who was a b l e t o show a c o m p l e t e h y p o t h a l a m o - n e u r o h y p o p h y s i a l s y s t e m i n t h e d o g f i s h S c y l l i u m s t e l i a r e by means o f s e r i a l s e c t i o n s , s t a i n e d w i t h G o m o r i ' s c h r o m h a e m a t o x y l i n - p h l o x i n s t a i n f o r n e u r o s e c r e t o r y g r a n u l e s . He d e m o n s t r a t e d a p r e o p t i c n u c l e u s , whose c e l l s c o n t a i n e d n e u r o s e c r e t o r y g r a n u l e s . A f i b e r t r a c t , a l s o l a d e n w i t h n e u r o s e c r e t o r y g r a n u l e s , l e d f r o m t h e p r e o p t i c n u c l e u s i n t o t h e p a r s i n t e r m e d i a o f t h e a d e n o h y p o p h y s i s , where t h e r e were a l s o c l e a r a c c u m u l a t i o n s o f n e u r o s e c r e t o r y m a t e r i a l . He t h e r e b y d e m o n s t r a t e d t h a t a p a r s n e r v o s a i s i n d e e d p r e s e n t i n S c y l l i u m . H owever, i t i s a d i f f u s e s t r u c t u r e , and c o n s i s t s o f t h e sum o f a l l t h e s e c r e t i o n - l a d e n n e r v e e n d i n g s o f t h e 17 p r e o p t i c o - h y p o p h y s i a l t r a c t , i n t i m a t e l y i n t e r m i n g l e d with the c e l l s of the pars i n t e r m e d i a . Together the pars nervosa and pars i n t e r m e d i a form the neurointermediate lobe (NIL) of the elasmobranch p i t u i t a r y gland (Perks, i 9 6 0 ) . In 1962, Braak found a s l i g h t l y m o d i f i e d form of neurohypophysial s t r u c t u r e i n the shark, Spinax n i g e r . He demonstrated the system by means of s e r i a l s e c t i o n s , s t a i n e d f o r n e u r o s e c r e t o r y m a t e r i a l by Gomori's al d e h y d e - f u c h s i n technique. Braak showed t h a t the l a r g e neurons of the p r e o p t i c nucleus were impregnated with powdery ne u r o s e c r e t o r y g r a n u l e s , and he was able to t r a c e the p r e o p t i c o - h y p o p h y s i a l t r a c t throughout i t s l e n g t h . He found that the pars nervosa of t h i s s p e c i e s , i n c o n t r a s t to that of S c y l l i u m , formed a r e l a t i v e l y t h i n t i s s u e l a y e r on the d o r s a l surface of the pars i n t e r m e d i a . A blood v e s s e l network separated the n e u r a l t i s s u e from the pars i n t e r m e d i a , and the s e c r e t i o n - l a d e n nerve endings of the p r e o p t i c o - h y p o p h y s i a l t r a c t terminated on the basement membrane of t h i s network. F i n g e r - l i k e e v a g i -n a t i o n s of the i n f u n d i b u l a r f l o o r and the u n d e r l y i n g pars nervosa p e n e t r a t e d the pars i n t e r m e d i a , and together they formed the neurointermediate lobe. Braak f u r t h e r confirmed the s t r u c t u r e of the hypothalamo-neurohypophysial system with a whole-mount s t a i n i n g and embedding technique. Whole b r a i n s of Spinax n i g e r were s e l e c t i v e l y s t a i n e d with Humberstone 1s V i c t o r i a Blue s o l u t i o n , a m o d i f i c a t i o n of Adams and Slope r ' s performic a c i d - a l c i a n 18 blue s t a i n f o r n e u r o s e c r e t i o n . The b r a i n s were then embedded i n t r a n s p a r e n t benzyl-benzoate m e t h y l - s a l i c y l a t e . By t h i s technique the e n t i r e morphology of the system could be observed at a glance and i t s s p a t i a l r e l a t i o n s h i p s a p p r e c i a t e d . Again the pars nervosa was found to be a d i s c r e t e s t r u c t u r e , although indented i n t o the pars i n t e r m e d i a . M e u r l i n g (1962) performed a d e t a i l e d h i s t o l o g i c a l study of the p i t u i t a r y of the spiny d o g f i s h Squalus a c a n t h i a s , one of the species i n v e s t i g a t e d i n the present study. In t h i s s p e c i es the s e c r e t i o n - l a d e n nerve endings were a l s o separated from the pars i n t e r m e d i a by a connective t i s s u e membrane, and were co n f i n e d to the i n f u n d i b u l a r f l o o r . This was i n agreement with the f i n d i n g s of S t e r z i and Baumgartner i n the same s p e c i e s . The pars nervosa of Squalus, l i k e t h a t of Spinax, sends t h i c k processes i n t o the pars i n t e r m e d i a , and these processes c o n s i s t of f o l d i n g s of the e n t i r e i n f u n -d i b u l a r f l o o r . Meurling d i d n o t i c e a small degree of nerve p e n e t r a t i o n from n e u r a l to i n t e r m e d i a t e t i s s u e , u s u a l l y i n p l a c e s where the s e p a r a t i n g connective t i s s u e membrane was absent. As the above account i l l u s t r a t e s , the pars nervosa component of the elasmobranch neurointermediate lobe may occur i n two main h i s t o l o g i c a l l y d i s c e r n a b l e forms ( M e u r l i n g , 1962). These are f i r s t l y the d i f f u s e type d e s c r i b e d by Hogben, de Beer, and Scharrer i n the skate (sp?) and i n 19 S c y l l i u m s t e l l a r e , and secondly the d i s c r e t e type observed by S t e r z i , Baumgartner, and Braak i n Squalus acanthias and Spinax n i g e r . The d i s c r e t e type of pars nervosa remains as a d i s t i n c t part of the b r a i n w a l l , which sends p r o j e c t i o n s i n t o the d o r s a l s urface of the pars intermedia but remains separated from i t by a l a r g e l y continuous membrane. Although some neur o s e c r e t o r y axons may pass through gaps i n the mem-brane, the i n t e r m i n g l i n g with the pars in t e r m e d i a i s s l i g h t (Meurling, 1962; 1967d). Other elasmobranchs with a pars nervosa of s i m i l a r form to that of Squalus ac anthias are Etmopterus spinax = Spinax n i g e r (Braak, 1962; Me u r l i n g , 1963), Squatina s q u a t i n a ( M e l l i n g e r , 1966), and the f o l l o w i n g sharks, which are considered to be r e l a t i v e l y p r i m i t i v e - Heptanchus, Hexanchus, Heterodontus = the Port Jackson shark, and Chlamydoselachus ( S t e n d e l l , 1913; 19l4; N o r r i s , 19^1; C o l b e r t , 1 9 6 l ) . In the d i f f u s e type of pars nervosa there i s no l i m i t i n g membrane and the nerve f i b e r s of the hypothalamo-hypo p h y s i a l t r a c t penetrate and i n t e r m i n g l e with the t i s s u e of the pars in t e r m e d i a (Meurling, 1962; 1967d). In S c y l l i u m  s t e l l a r e , S c y l l i u m c a n i c u l a and Must elus must elus there i s a strong i n t e r m i n g l i n g of ne u r o s e c r e t o r y and intermedia compo-nents, but a d i s t i n c t pars nervosa l a y e r may s t i l l be recog-n i z e d i n the i n f u n d i b u l a r f l o o r ( S c h a r r e r , 1952; Bargmann, 1955; M e u r l i n g , 1962; 1967a; Perks, 1969). In P r i s t i u r u s  melanostomus and i n the skates and rays (Dasyatis marinus, 20 Trygon p a s t i n a c a = D a s y a t i s p a s t i n a c a , Torpedo o c e l l a t a , Raia bat i s , R. oxyrhynchus , R_. f u l l o n i c a , R. r a d i a t a , and R_. c l a v a t a ) there i s complete f u s i o n between n e u r a l and i n t e r m e d i a t e elements and one cannot d i s c e r n a pars nervosa as a d i s c r e t e m orphological e n t i t y (Bargmann, 1955; M e u r l i n g , 1962; 1967a; 1970; Polenov and Belenky, 1965; M e l l i n g e r , 1966; Perks, 1969). Although the d i f f u s e and d i s c r e t e types of pars nervosa may be found i n such species as the skates (ex. Raia  c l a v a t a ) and the d o g f i s h Squalus ac a n t h i as, r e s p e c t i v e l y , other species may show some degree of intermediate form i n t h e i r s t r u c t u r e s . However, p h y s i o l o g i c a l s t u d i e s might w e l l concentrate on the two extreme types mentioned here. 2. The Nature of the Elasmobranch Neurohypophysial  P r i n c i p l e s a. Evidence f o r neurohypophysial a c t i v i t y i n the  elasmobranch p i t u i t a r y The a c t i v i t y of elasmobranch neurohypophysial e x t r a c t s , as measured by standard bioassay techniques, i s very low. Therefore the demonstration of o x y t o c i c and a n t i d i u r e t i c p r i n c i p l e s i n these f i s h was e s p e c i a l l y d i f f i c u l t . In h i s remarkable pioneer work, He r r i n g (1908a; i908.b) was unable to f i n d t h a t p i t u i t a r y e x t r a c t s of the skate, Rai a  b a t i s , had any e f f e c t on the blood pressure or u r i n e flow of a n a e s t h e t i z e d c a t s , and he concluded that the a c t i v e p r i n c i p l e s found i n the pars nervosa and pars intermedia of mammals, 21 b i r d s , and t e l e o s t s were not present i n the elasmobranchs. However i n 1913 he found that a Rai a bat i s e x t r a c t caused an immediate and w e l l marked m i l k - e j e c t i o n response i n the l a c t a t i n g c a t . Later (1915) he observed that p i t u i t a r y -e x t r a c t s of the skate, Rai a c l a v a t a, caused c o n t r a c t i o n of the i s o l a t e d v i r g i n r a t u t e r u s . However, he a t t r i b u t e d t h i s a c t i v i t y to the chromophobe c e l l s of the pars in t e r m e d i a t i s s u e . Other b i o l o g i c a l a c t i v i t i e s c h a r a c t e r i s t i c of neuro-h y p o p h y s i a l e x t r a c t s were g r a d u a l l y a t t r i b u t e d to the elasmo-branch p i t u i t a r y . Hogben (1925) found an avine (duck) blood depressor response to skate (sp?) e x t r a c t s ; and Hogben and de Beer (1925) demonstrated a c o n t r a c t i o n of the guinea p i g u t e r u s , again using skate (sp?) p i t u i t a r y e x t r a c t . L a t e r , H e l l e r (l9^1a) showed that e x t r a c t s of both skate (sp?) and d o g f i s h (sp?) p i t u i t a r y exerted an a n t i d i u r e t i c e f f e c t i n the r a b b i t . On the other hand, Waring and Landgrebe (1950) f a i l e d i n "exhaustive" e f f o r t s to demonstrate r a t vasopressor or guinea p i g uterus responses to elasmobranch e x t r a c t s , and a s c r i b e d previous workers' f i n d i n g s to the presence of h i s t a -mine. However, as p o i n t e d out by Hogben and de Beer (1925), histamine a c t u a l l y r e l a x e s the r a t u t e r u s , and so i t could not be r e s p o n s i b l e f o r the c o n t r a c t i o n s observed by H e r r i n g i n the r a t . 22 In 1959 Maetz et a l . not only confirmed o x y t o c i c a c t i v i t y ( c o n t r a c t i o n of the i s o l a t e d r a t uterus) i n the elasmobranchs Hexanchus g r 1 s eus and S c y l i o r h i n u s can 1 c u i u s , but a l s o demonstrated yet another p r o p e r t y , a n a t r i f e r i c e f f e c t . They observed that p i t u i t a r y e x t r a c t s of these species brought about the a c t i v e t r a n s p o r t of Wa + across the s k i n of the amphibian Rana e s c u l e n t a , an e f f e c t a l s o e x h i b i t e d by very low c o n c e n t r a t i o n s of t e l e o s t and amphibian neuro-hypop h y s i a l e x t r a c t s . In the same year, Sawyer (1959) a l s o confirmed the presence of neurohypophysial a c t i v i t y i n the p i t u i t a r y of the d o g f i s h , Squalus a c a n t h i a s . During the same p e r i o d of r e s e a r c h , Perks et a l . (i960; Perks and Dodd, 1963a; 1963b) subjected n e u r o i n t e r -mediate l o b e , saccus v a s c u l o s u s , hypothalamus, " i n d i f f e r e n t b r a i n " , and v e n t r a l lobe e x t r a c t s of s e v e r a l elasmobranch species (Squalus a c a n t h i a s , S c y l i o r h i n u s c a n i c u l u s , Raia  c l a v a t a , and Raia b a t i s ) to a s e r i e s of b i o a s s a y s , which i n c l u d e d the i s o l a t e d r a t u t e r u s , r a b b i t m i l k - e j e c t i o n , r a t vasopressor, and r a t a n t i d i u r e t i c e s t i m a t i o n s . They demon-s t r a t e d that r a t u t e r u s , milk-e j.ection, and a n t i d i u r e t i c a c t i v i t i e s were indeed present i n a l l four s p e c i e s , and furthermore that these a c t i v i t i e s were p r e f e r e n t i a l l y l o c a -ted i n the neurointermediate lobe. No vasopressor a c t i v i t y was detected. In f a c t , a s l i g h t vasodepressor e f f e c t was noted, but Her r i n g (1915) had found t h i s e f f e c t to be due to contaminants which could be removed from the e x t r a c t s by treatment with absolute a l c o h o l . C o n f i r m a t i o n that these neurointermediate lobe a c t i v i t i e s were i n f a c t due to neurohypophysial p r i n c i p l e s l a y i n the ob s e r v a t i o n s that they were destroyed by t r e a t -ment with sodium t h i o g l y c o l l a t e , which reduces the S-S bond of c y s t i n e (van Dyke, 1942), with sodium hydroxide and by u l t r a v i o l e t l i g h t (Dudley, 1920). It i s important t h a t h i s t a m i n e , suggested by Waring and Landgrebe to be respon-s i b l e f o r the elasmobranch "neurohypophysial" a c t i v i t y , i s r e s i s t a n t to sodium hydroxide treatment (Dudley, 1920). Perks and Dodd found that the m i l k - e j e c t i o n , o x y t o c i c , and a n t i d i u r e t i c p o t e n c i e s of the elasmobranch n e u r o i n t e r -mediate lobe e x t r a c t s , when they were assayed against mammalian p r i n c i p l e s , were g e n e r a l l y i n the r a t i o 3:1:0.05. This a c t i v i t y p r o f i l e suggested that the elasmobranchs do not produce a v a s o p r e s s o r - a n t i d i u r e t i c p r i n c i p l e , but that they do e l a b o r a t e an o x y t o c i c p r i n c i p l e which i s d i f f e r e n t from o x y t o c i n i t s e l f . T h i s p r i n c i p l e would have a small i n t r i n s i c a n t i d i u r e t i c a c t i v i t y , and i t s m i l k - e j e c t i o n a c t i v i t y would be g r e a t e r than i t s o x y t o c i c ( r a t uterus) e f f e c t . The elasmobranch a c t i v i t y p r o f i l e was extended to four f u r t h e r s p e c i e s , D a s y a t i s sabina ( s t i n g r a y ) , Sphyrna. mokarran (hammerhead shark) , E u l a n i a m i l b e r t i (brown shark) , and Carcharinus leucas ( b u l l s h a r k ) , and was confirmed f o r 2k the d o g f i s h Squalus acanthias (Perks, 1 9 6 6 ) . Perks a l s o found that the o x y t o c i c agent from these species behaved ch r o m a t o g r a p h i c a l l y l i k e o x y t o c i n , which was i n agreement with a s i m i l a r i t y between t h e i r molecules. Sawyer et a l . ( 1 9 6 1 ) confirmed and extended the phar-m a c o l o g i c a l p r o f i l e of the o x y t o c i c agent of Squalus a c a n t h i a s . By a d m i n i s t e r i n g l a r g e doses they were able to demonstrate a weak r a t vasopressor a c t i v i t y , although t h i s was p a r t i a l l y obscured by the r a t vasodepressor e f f e c t noted by H e r r i n g ( 1 9 1 5 ) and by Perks and Dodd ( 1 9 6 3 a ) . They showed that Squalus neurointermediate lobe e x t r a c t s caused c o n t r a c t i o n of the hen o v i d u c t , brought about water t r a n s p o r t across the i s o l a t e d f r o g b l a d d e r , and exerted a fowl vasodepressor e f f e c t . In a d d i t i o n they measured the o x y t o c i c a c t i v i t y of the Squalus e x t r a c t s a f t e r the a d d i t i o n of magnesium ions to the medium i n which the r a t u t e r i n e horn was suspended. The presence of magnesium ions augments the response of the r a t uterus to neurohypophysial p e p t i d e s , but the i n c r e a s e i s g r e a t e r f o r o x y t o c i n analogues than f o r o x y t o c i n i t s e l f (Munsick, i 9 6 0 ) . Therefore the potency of an analogue measured against an o x y t o c i n standard i n the presence of magnesium i s gr e a t e r than i t s potency when the assay i s performed without magnesium. The apparent i n c r e a s e i n potency due to the presence of magnesium ions may be d e f i n e d as f o l l o w s (Sawyer, 1 9 6 7 ) : 25 Magnesium ( M g + + ) p o t e n t i a t i o n = R = Mg a c t i v i t y on r a t u t e r u s i n p r e s e n c e o f 0 . 5mM Mg + + a c t i v i t y on r a t u t e r u s i n a b s e n c e o f M g + + The R„, o f t h e two S q u a l u s e x t r a c t s i n v e s t i g a t e d by Sawyer Mg et a l . (1961) were 1.27 and 2.03. A magnesium p o t e n t i a t i o n o f a p p r o x i m a t e l y 2 f o r t h i s s p e c i e s was l a t e r c o n f i r m e d by Sawyer (R = 2.6; 1967) and by S w i a t k i e w i c z (R = 1.2 - 3.2; JYL g JYl g 1968). b. E v i d e n c e s u p p o r t i n g t h e e x i s t e n c e o f an  e l a s m o b r a n c h a n t i d i u r e t i c p r i n c i p l e W h i l e most v e r t e b r a t e s p o s s e s s an a n t i d i u r e t i c -v a s o p r e s s o r p e p t i d e as w e l l as an o x y t o c i c p r i n c i p l e , t h e r e s u l t s o f t h e e a r l y i n v e s t i g a t i o n s d i d not s u p p o r t t h e p r e s e n c e o f s u c h an a n t i d i u r e t i c f r a c t i o n among t h e e l a s m o -b r a n c h s ( P e r k s e_t a l . , I960; P e r k s and Dodd, 1963a; Sawyer et a l . , 1959; H e l l e r and P i c k e r i n g , 1961) and i t a p p e a r e d t h a t e l a s m o b r a n c h s e l a b o r a t e d an o x y t o c i c f r a c t i o n o n l y . However l a t e r work, p u b l i s h e d d u r i n g t h e c o u r s e o f t h e p r e s e n t s t u d y , and o u t l i n e d b e l o w , i n d i c a t e d t h a t an a r g i n i n e v a s o t o c i n - l i k e p r i n c i p l e was p r e s e n t , a l t h o u g h i n v e r y s m a l l amount s. A c h e r and h i s group ( A c h e r et a l . , 1965; 1967; Chauvet et a l . , 1965) f o u n d i n f o u r s p e c i e s o f s k a t e ( R a i a c l a v a t a , R. b a t i s , R. naevus , and R_. f u l l o n i c a ) t h a t i n a d d i t i o n t o t h e main o x y t o c i c f r a c t i o n t h e r e was a s e c o n d v e r y s m a l l f r a c t i o n w h i c h p o s s e s s e d p r e s s o r a c t i v i t y e q u a l t o , or 26 g r e a t e r than, i t s o x y t o c i c a c t i v i t y , and which had hydro-osmotic a c t i v i t y i n a d d i t i o n . This f r a c t i o n "behaved l i k e a r g i n i n e v a s o t o c i n "both during e l u t i o n chromatography from Amberlite CG-50 columns and during paper chromatoelectro-p h o r e s i s . Sawyer ( l ° 6 5 , I 9 6 7 ) found that the dogfish, Squalus  ac a n t h i as, e l a b o r a t e d a second small o x y t o c i c f r a c t i o n which possessed only about 1 . 2 - l.k% of the o x y t o c i c a c t i v i t y of the main f r a c t i o n , but which accounted f o r almost a l l of the t o t a l a n t i d i u r e t i c and f r o g - b l a d d e r a c t i v i t y . This small f r a c t i o n could be separated from the main a c t i v i t y e i t h e r by e l u t i o n chromatography on CM c e l l u l o s e or on CM Sephadex columns, or by paper chromatography. In both cases i t s behavior was the same as that of a r g i n i n e v a s o t o c i n . The r e l a -t i v e p h a r m a c o l o g i c a l a c t i v i t i e s of t h i s f r a c t i o n ( a n t i d i u r e t i c a c t i v i t y , f r o g - b l a d d e r a c t i v i t y , and r a t uterus a c t i v i t y i n the presence or absence of Mg + +) a l s o matched those of synthe-t i c a r g i n i n e v a s o t o c i n . c. Evidence supporting the e x i s t e n c e of s e v e r a l  elasmobranch o x y t o c i c p r i n c i p l e s Soon a f t e r i t had been e s t a b l i s h e d that the elasmo-branch o x y t o c i c agent was not o x y t o c i n i t s e l f , i t began to appear that i t represented not one but s e v e r a l n e u t r a l p r i n c i p l e s . However, i t was not c l e a r whether each elasmo-branch species e l a b o r a t e d a s i n g l e n e u t r a l p r i n c i p l e which v a r i e d between s p e c i e s , or whether each species produced a mixture of p e p t i d e s , and that the p r o p o r t i o n s of the peptides 2.7 i n the mixture v a r i e d . The d i f f e r e n t p r i n c i p l e s could he d i s t i n g u i s h e d by t h e i r chromatographic and pharmacological c h a r a c t e r i s t i c s i n the f o l l o w i n g three ways: i . By d i f f e r e n c e s i n r e l a t i v e m i l k - e j e c t i o n a c t i v i t i e s . i i . By d i f f e r e n c e s i n magnesium p o t e n t i a t i o n , i i i . By d i f f e r e n c e s i n chromatographic behavior. Each of these methods of d i f f e r e n t i a t i o n w i l l be d i s c u s s e d s e p a r a t e l y , below. i . D i f f e r e n c e s between the o x y t o c i c p r i n c i -p l e s , based on r e l a t i v e m i l k - e j e c t i o n  act i v i t i e s, As mentioned p r e v i o u s l y , the general r a t i o of milk-e j e c t i o n a c t i v i t y to r a t uterus a c t i v i t y of the elasmobranch neurohypophysial e x t r a c t s i s approximately 3:1. This has been found f o r at l e a s t eight species (Perks and Dodd, 1963a; Perks, 1966; Sawyer e_t al_. , 196l; Sawyer, 1967). S u r p r i s i n g l y , upon p u r i f y i n g the o x y t o c i c p r i n c i p l e of the ray Raia o c e l l a t a , Perks and Sawyer (1965) found that the r e l a t i v e m i l k - e j e c t i o n a c t i v i t y of both the crude e x t r a c t and the p u r i f i e d p r i n c i p l e was much higher than expected. The r a t i o of m i l k - e j e c t i o n to r a t uterus a c t i v i t y of the crude e x t r a c t was 10.3±1.1 and that of the p u r i f i e d peptide was 8.5*1.0. These two r a t i o s were not s i g n i f i c a n t l y d i f f e r e n t , but c o n t r a s t e d with those of a l l species p r e v i o u s l y i n v e s t i g a t e d . The magnesium poten-t i a t i o n , and the r a t i o of a n t i d i u r e t i c to r a t uterus a c t i v i t y , 28 were of the expected magnitude, hut the r e l a t i v e f r o g -bladder a c t i v i t y of the p u r i f i e d R . o c e l l a t a p r i n c i p l e was much lower than that found p r e v i o u s l y f o r crude Squalus  acanthias e x t r a c t s (Sawyer e_t_ a l . , 1961) . However , t h i s d i f f e r e n c e may simply have r e f l e c t e d the presence of the v a s o t o c i n - l i k e p r i n c i p l e i n the crude Squalus p r e p a r a t i o n . I f t h i s i s so, then the only c l e a r way i n which the R. o c e l l a t a p r i n c i p l e d i f f e r e d from that of other elasmobranchs was i n i t s high m i l k - e j e c t i o n potency. This d i f f e r e n c e l e d to the c l a s s i f i c a t i o n (Sawyer, 1964) of elasmobranch o x y t o c i c p r i n c i p l e s i n t o EOP I (elasmobranch o x y t o c i c p r i n c i p l e I) and EOP II (elasmobranch o x y t o c i c p r i n c i p l e I I ) . EOP I, found i n the m a j o r i t y of elasmobranchs, had a r a t i o of m i l k - e j e c t i o n to r a t uterus a c t i v i t y of approximately 3. EOP I I , o c c u r r i n g i n R . o c e l l a t a , had a r a t i o of from 8 to 10 (see Table I I I ) . The R . o c e l l a t a p u r i f i e d • p e p t i d e , EOP I I , was sub-j e c t e d to amino a c i d a n a l y s i s and found to c o n s i s t of c y s t i n e , t y r o s i n e , s e r i n e , glutamic a c i d , a s p a r t i c a c i d , p r o l i n e , i s o -l e u c i n e , and g l y c i n e i n approximately equimolar amounts (Perks and Sawyer, 1965). i i . D i f f e r e n c e s between the o x y t o c i c p r i n -c i p l e s , based on magnesium p o t e n t i a t i o n s Acher e_t al_. (1965) p u r i f i e d the major a c t i v e f r a c t i o n of the thornback ray, Raia c l a v a t a , and by enzymic degrada-t i o n determined i t s amino a c i d sequence to be that of 4-serine , 29 8-glutamine o x y t o c i n , which they c a l l e d " g l u m i t o c i n " . . The same amino a c i d composition and chromatoel.ectrophoret.ic behavior was found f o r the major a c t i v e f r a c t i o n of R. b a t i s (Chauvet et_ al_. , 1965) , R_. naevus and R. f u l l o n i c a (Acher et_ a l . , 1 9 6 7 ) , which suggested that they a l s o contained glumi-t o c i n . G l u m i t o c i n has the same amino a c i d composition as EOP I I , but d i f f e r s i n i t s magnesium p o t e n t i a t i o n . The RMg of g l u m i t o c i n from R. c l a v a t a and R_. b a t i s was found t o be 10, the hig h e s t value r e p o r t e d at that time f o r any n a t u r a l l y o c c u r r i n g o x y t o c i n analogue. In c o n t r a s t , a lower p o t e n t i a t i o n of approximately 2 had been found f o r EOP I I , and a l s o f o r EOP I. Another of the p o s t u l a t e d elasmobranch p r i n c i p l e s , Ej_ (see S e c t i o n i i i , below) had been c h a r a c t e r i z e d by a high magnesium p o t e n t i a t i o n value of 6 i n some s p e c i e s , although the value was lower i n s e v e r a l other s p e c i e s , and s i m i l a r to that found f o r EOP I and EOP I I . C l e a r l y , at t h i s time c o n s i d e r a t i o n s of the magnesium p o t e n t i a t i o n s of elasmobranch neurohypophysial p r i n c i p l e s were complex and d i f f i c u l t to i n t e r p r e t . i i i • D i f f e r e n c e s between the o x y t o c i c p r i n c i p l e s , based on chromatographic  behavi or (1) The r e s o l u t i o n of two o x y t o c i c p r i n c i p l e s w i t h i n a single' species Descending paper chromatograms of crude e x t r a c t s of two d o g f i s h , Squalus acanthias and S c y l i o r h i n u s can 1 c u i u s , 30 developed i n n-butanol: a c e t i c a c i d : water = U:l:5 were found to y i e l d two areas of o x y t o c i c a c t i v i t y ( H e l l e r and P i c k e r i n g , 1961). During the time of the present study, t h i s o b s e r v a t i o n was extended to an a d d i t i o n a l nine species ( H e l l e r and Roy, 1965a; 1965b; 1967; Roy, 1969). One of the f r a c t i o n s ran with an R-p of approximately 0.5 to 0.8, the same R as o x y t o c i n i n t h i s s o l v e n t system. The other f r a c t i o n ran more s l o w l y , with an R^ of approximately 0.3 to 0.5. The two f r a c t i o n s were designated as E-^, the slow-moving moiety, and as Eg, the fast-moving p r i n c i p l e . Both E^ and Eg were l a b i l e to sodium t h i o g l y c o l l a t e , an i n d i c a t i o n that both f r a c t i o n s r e p r e s e n t e d true neurohypophysial p r i n c i p l e s . E^ and Eg have been f u r t h e r d i s t i n g u i s h e d by t h e i r p h a rmacological p r o p e r t i e s , mainly by t h e i r magnesium potent-i a t i o n . On t h i s b a s i s the species s t u d i e d may be d i v i d e d i n t o three groups, which w i l l be d i s c u s s e d i n t u r n : (a) Species i n which E^ and Eg have d i f f e r e n t magnesium p o t e n t i a t i o n s In t h i s case the fast-moving Eg was c h a r a c t e r i z e d by a p o t e n t i a t i o n of approximately 2 and the slow-moving E^ showed a higher value of approximately 6. Such a d i f f e r e n c e was found i n the spotted d o g f i s h S c y l i o r h i n u s c a n i c u i u s ( H e l l e r and Roy, 1965b; Roy, 1969), the g u i t a r f i s h 'Rhino -b a t i s r h i n o b a t i s (Roy, 1969) and the thornback ray Raia  c l a v a t a ( H e l l e r and Roy, 1965a; Roy, 1969). 31 The p h a r m a c o l o g i c a l d i f f e r e n c e between E^ and E^ was f u r t h e r s u b s t a n t i a t e d by d i f f e r e n c e s i n n a t r i f e r i c and a n t i -d i u r e t i c potency. N a t r i f e r i c a c t i v i t y was found i n the f a s t -moving f r a c t i o n , E^ , of S c y l i o r h i n u s and R a i a , but none could be d e t e c t e d i n the slow-moving f r a c t i o n ( H e l l e r and Roy, 1965a; Roy, 1969). The a n t i d i u r e t i c a c t i v i t y of r e l a t i v e to i t s o x y t o c i c a c t i v i t y was g r e a t e r than that of E^ (Roy, 1969). However, since the values given were only approxima-t i o n s based on a few assay responses, the s i g n i f i c a n c e of the r e s u l t i s d i f f i c u l t to assess. No s i g n i f i c a n t d i f f e r e n c e was found i n the guinea p i g m i l k - e j e c t i o n a c t i v i t i e s of the two f r a c t i o n s r e l a t i v e to t h e i r o x y t o c i c a c t i v i t i e s (Roy, I969). (b) Species i n which E^ and Eg have s i m i l a r magnesium p o t e n t i a t i o n s The p o t e n t i a t i o n s of both f r a c t i o n s were approximately 2. T h i s was found i n the b u l l shark Carcharinus leucas (Roy, 1969) and i n the lemon shark Negaprion b r e v i r o s t r i s ( H e l l e r and Roy, 1965b; Roy, 1969). In a d d i t i o n to the s i m i l a r i t y i n t h e i r magnesium p o t e n t i a t i o n s , the two f r a c t i o n s showed r e l a t i v e guinea p i g m i l k - e j e c t i o n a c t i v i t i e s which were not s i g n i f i c a n t l y d i f f e r -ent. The n a t r i f e r i c a c t i v i t i e s were not t e s t e d . In both s p e c i e s , only the r e l a t i v e a n t i d i u r e t i c a c t i v i t y of the slow f r a c t i o n was found to be g r e a t e r than that of the f a s t -moving moiety (Roy, 1969). Again, however, these were only approximate assays, and the r e s u l t s may not be s i g n i f i c a n t . 32 Therefore i n the case of Carcharinus and Negaprion the only-c h a r a c t e r i s t i c which d i s t i n g u i s h e s c l e a r l y "between E^ and E 2 i s t h e i r chromatographic m o b i l i t y . (c) Species i n which n e i t h e r E^ nor Eg i s p o t e n t i a t e d by magnesium ions Th i s i n c l u d e d a p r i m i t i v e elasmobranch, the cow shark Hexanchus g r i s e u s , as w e l l as three h o l o c e p h a l i a n s , the r a b b i t f i s h Chimaera monstrosa, the r a t f i s h Hydrolagus c o l l e i , and Callorhynchus sp. ( H e l l e r and Roy, 1965b; 1967; Roy, 1969) . The high a n t i d i u r e t i c a c t i v i t y of E^ i n Hexanchus has been a t t r i b u t e d to contamination of the slow f r a c t i o n by a r g i n i n e v a s o t o c i n (Roy, 1969) and the n a t r i f e r i c and guinea p i g m i l k - e j e c t i o n a c t i v i t i e s of the two f r a c t i o n s i n these species have not been i n v e s t i g a t e d . Again i n t h i s case the only c l e a r d i f f e r e n c e between E and Eg i s i n t h e i r chromatographic m o b i l i t y . ( 2 ) The r e s o l u t i o n of a s i n g l e o x y t o c i c  f r a c t i o n w i t h i n each species The chromatographic d i f f e r e n t i a t i o n of the elasmo-branch o x y t o c i c agent i n t o two f r a c t i o n s , E-^  and Eg, by H e l l e r and h i s group has not been observed by other workers. Perks (1966) s u b j e c t e d f i v e s p e c i e s , the d o g f i s h Squalus  a c a n t h i a s , the s t i n g r a y D a s y a t i s s a b i n a , the hammerhead shark Sphyrna mokarran, the brown shark E u l a n i a m i l b e r t i , and the b u l l shark Carcharinus leucas , to chromatography on both Whatman 3MM paper and gl a s s paper impregnated with s i l i c i c a c i d . A v a r i e t y of sol v e n t systems was used , Table I I I D i s t i n g u i s h i n g C h a r a c t e r i s t i c s of the Elasmobranch Oxytocic P r i n c i p l e s Pr i n c i p l e Species Paper chromat ography Rp a Pharmacolog RME ; i c a l r a t i o s RMg Referenc EOP I Squalus acanthias 0.5-0.8 1.5-2.6 1.2-3.2 1,2,3,4,5 S c y l i o r h i n u s c a n i c u l u s - 3.0,4.5 - 2 Raia c l a v a t a - 3.5 - 2 Raia bat i s - 4.5 - 2 Dasyatis sabina 0.6-0.8 3.4 - 5 Sphyrna mokarran 0.4-0. 6 2.6 - 5 E u l a n i a mi-lberti 0.5-0.7 3.8 - 5 Carcharinus leucas 0.5-0.7 C 4.2 - 5 EOP II Raia o c e l l a t a - 8.5 ,10.3 2.6 3 Gl u m i t o c i n Raia c l a v a t a _ 10 8,9 Raia b a t i s - - 10 9 Raia naevus - - 10 10 Raia f u l l o n i c a - - 10 10 E 1 ( R M g = 6 ) S c y l i o r h i n u s c a n i c u l u s 0.25-0.50° — 5.5,6.3 11,13 ,14 •Raia c l a v a t a 0.04-0.40 c - 6.1 12 ,14 Rhinobatis r h i n o b a t i s 0.35-0.60 c - 5.1 lk E!(R M g=2) Carcharinus leucas 0.30-0.50° _ 1.8 lk Negaprion b r e v i r o s t r i s 0.35-0.45 - 2.9,3.0 13 ,14 E 1 ( R M g = ? ) Squatina squatina 0.10-0.30 - - 14 Squalus acanthias 0.35-0.45 - - 11 Table I I I continued P r i n c i p l e Species Paper chromatography R F a Pharmacolog r i c a l r a t i o s Reference* 5 R M E RMg E 1 ( R M g = l ) Hexanchus g r i s e u s 0.10-0.20 - 0.95 ,1.3 14 E 2^ RMg = 2^ S c y l i o r h i n u s c a n i c u l u s Raia c l a v a t a Rhinobatis r h i n o b a t i s Carcharinus leucas Wegaprion b r e v i r o s t r i s Squatina squatina 0.50-0.90 c 0.35-0.80C 0 . 7 0 - 0 . 9 0 ° 0 . 6 5 - 0 . 9 0 ° 0.5 - 0 . 8 ° 0.45-0.90 - 2.5 2.1 2.0 1.9 2.2,2 .6 2.0 11 ,13 ,14 12 ,14 lk lk 13 ,14 14 E 2(RMg=?) Squalus acanthias 0.5 -0.6 - - l l E 2 ( R M g = 1 ) Hexanchus g r i s e u s 0 . 4 5 - 0 . 9 0 ° - 1.0,0.85 14 A b b r e v i a t i o n s used: Rp> = the r a t i o of the dis t a n c e from the o r i g i n to the l o c a t i o n of the o x y t o c i c p r i n c i p l e / d i s t a n c e from the o r i g i n to the solvent f r o n t , on a paper chromatogram developed i n n-butanol: a c e t i c acid:water = 4:1:5. Rjy[g = r a t i o of r a b b i t m i l k - e j e c t i o n a c t i v i t y / a c t i v i t y on the r a t uterus ( Mg + + absent). R^g = magnesium poten-t i a t i o n (see t e x t , page 25). ^References: 1: Sawyer et_ a l . (1961), 2: Perks and Dodd (1963a), 3: Perks and Sawyer ( 1 9 6 5 ) , 4: Sawyer ( 1965T, 5: Perks (1966), 6: Sawyer (1967), 7: Swiatkiewicz (1968) , 8: Acher et_ a l . (1965), 9: Chauvet et a l . (1965) , 10: Acher et a l . (1967) , 1 1 : H e l l e r and P i c k e r i n g T T 9 6 1 ) , 12: H e l l e r and Roy"Tl965a), 13: H e l l e r and Roy (1965b), 14: Roy (1969). c E n t i r e range, more than one chromatogram. 35 i n c l u d i n g the n - h u t a n o l : a c e t i c acid:water = 4:1:5 system of H e l l e r , P i c k e r i n g , and Roy. In a l l cases the o x y t o c i c a c t i v i t y ran as one f r a c t i o n , with an R-p s i m i l a r to that of o x y t o c i n , or to the f a s t - r u n n i n g E p r i n c i p l e of H e l l e r . I t i s i n t e r -2 e s t i n g that two of the s p e c i e s , Squalus acanthias and Carcharinus leucas , were used "by both Perks and H e l l e r with d i f f e r e n t r e s u l t s . It may be noted that Perks had chroma-togrammed crude neurointermediate lobe e x t r a c t s while H e l l e r and h i s co-workers d e p r o t e i n i z e d at l e a s t some of t h e i r e x t r a c t s with t r i c h l o r a c e t i c a c i d . A d s o r p t i o n of the a c t i v e p r i n c i p l e s onto p r o t e i n may have prevented Perks' e a r l i e r e x t r a c t s from r e s o l v i n g . However i n l a t e r experiments Perks (p e r s o n a l communication, 1970) was s t i l l unable to achieve r e s o l u t i o n of Squalus e x t r a c t s i n t o slow- and fast-moving components, even a f t e r p r e l i m i n a r y treatment of the e x t r a c t s with t r i c h l o r a c e t i c a c i d (TCA). Swiatkiewicz (1968) ran repeated chromatograms of Squalus ac a n t h i as e x t r a c t s , with s t e a d i l y i n c r e a s i n g l o a d s , i n the sol v e n t system n - b u t a n o l : a c e t i c acid:water = 4:1:5; each time he found only one a c t i v e o x y t o c i c f r a c t i o n . Again, the magnesium p o t e n t i a t i o n was always approximately 2 (R^ =1.2-3.2 ) . Although h i s e x t r a c t s had not been t r e a t e d with TCA, some of them had been d e p r o t e i n i z e d by g e l f i l t r a t i o n through Sephadex G-15, and t h e r e f o r e they were q u a n t i t a t i v e l y comparable to H e l l e r ' s e x t r a c t s i n t h e i r t o t a l p r o t e i n content. 36 U s i n g t h e same s o l v e n t s y s t e m , Sawyer (1967) r a n p a p e r chromatograms o f t h e a c t i v e p r i n c i p l e s o f t h e same s p e c i e s , S q u a l u s ac a n t h i a s , a t t h r e e s t a g e s i n t h e i r p u r i f i -c a t i o n . These t h r e e s t a g e s were: 1) a f t e r d e p r o t e i n i z a t i o n by t r e a t m e n t w i t h TCA 2) a f t e r g e l f i l t r a t i o n t h r o u g h Sephadex G-25 3) a f t e r i o n exchange c h r o m a t o g r a p h y on CM Sephadex, a c a t i o n e x c h a n g e r . The T C A - t r e a t e d and Sephadex G - 2 5 - p u r i f i e d e x t r a c t y i e l d e d a major o x y t o c i c f r a c t i o n a t R-p 0.5-0.8 or 0.5-0.9, and a much s m a l l e r a r g i n i n e v a s o t o c i n - l i k e f r a c t i o n ( s e e s e c t i o n b above, page 23) at R-p 0.3-0.4. F o l l o w i n g e l u t i o n o f t h e p r i n c i p l e s f r o m t h e CM Sephadex, t h i s s m a l l f r a c t i o n had been s e p a r a t e d away, and a l l t h e r e m a i n i n g o x y t o c i c a c t i v i t y was f o u n d t o r u n on p a p e r i n one f r a c t i o n , a g a i n at R-p 0.5-0.8. T h i s a c t i v i t y may w e l l have c o r r e s p o n d e d t o t h e f a s t - r u n n i n g p r i n c i p l e , E^ , b u t a g a i n t h e r e was no e v i d e n c e f o r t h e p r e s e n c e o f t h e E^ m o i e t y . O n l y one o x y t o c i n - l i k e p r i n c i p l e was f o u n d i n t h e s p e c i e s w h i c h e l a b o r a t e e i t h e r EOP I I or g l u m i t o c i n . In t h e p u r i f i c a t i o n and amino a c i d a n a l y s i s o f t h e R a i a o c e l l a t a p r i n c i p l e , EOP I I ( s e e s e c t i o n c, i , page 25), P e r k s and Sawyer (1965) f o u n d e v i d e n c e o f o n l y one p e p t i d e , and A c h e r and h i s c o - w o r k e r s (1965; 1967) f o u n d o n l y one o x y t o c i n - l i k e p e p t i d e ( s e e s e c t i o n c, i i , page 26), w i t h a t r a c e o f a v a s o t o c i n - l i k e p r i n c i p l e ( s e e s e c t i o n b, page 23), i n t h e i r 37 p u r i f i c a t i o n and analysis, of Rai a c l a v a t a , R.. bat i s , R_. f u l l o n i c a , and R. naevus. I t i s noteworthy th.at' R. c l a v a t a i s one of the species i n which H e l l e r found a d i v i s i o n i n t o E-^  and Eg, where these, p r i n c i p l e s showed magnesium p o t e n t i a t i o n s of 6 and 2, r e s p e c t i v e l y . There i s no immediate e x p l a n a t i o n f o r the i n c o n -s i s t e n c y i n the appearance of the p r i n c i p l e s E-^  and Eg. I t may be that t h e i r occurrence v a r i e s with season or with other f a c t o r s , not yet understood (Perks, 1 Q66). There i s a p o s s i b i l i t y that E^ might be a r g i n i n e v a s o t o c i n , which a l s o runs slowly on paper i n the s o l v e n t system used; however, t h i s i s o f f s e t by the f o l l o w i n g c o n s i d e r a t i o n s : 1. In chromatograms of some of the s p e c i e s (Scy-l i o r h i n u s c a n i c u l u s , Carcharinus l e u c a s , Negaprion b r e v i r o s t r i s , and Hydrolagus c o l l e i ) a t h i r d d i s c r e t e o x y t o c i c area of very l i t t l e a c t i v i t y was found running even more slowly than Ej_ (Roy, 1969). 2. N e i t h e r of the two r e l a t i v e l y f a s t e r - r u n n i n g components of S c y l i o r h i n u s ( t h a t i s , Ej_ and E^) was i n a c t i v a t e d by t r y p s i n , although s y n t h e t i c a r g i n i n e v a s o t o c i n was completely i n a c t i v a t e d (Roy, 1969). 3. The r a t i o of a n t i d i u r e t i c a c t i v i t y to rat uterus a c t i v i t y i s g r e a t e r f o r a r g i n i n e v a s o t o c i n that f o r the slow-running E^ p r i n c i p l e (Roy, 1969). It i s c l e a r that the number and nature of the elasmo-branch o x y t o c i n - l i k e p r i n c i p l e s i s u n c e r t a i n and confused. 38 EOP I, EOP I I , and g l u m i t o c i n represent d i f f e r e n c e s between s p e c i e s , while E± and E 2 , on the other hand, are both produced w i t h i n a s i n g l e s p e c i e s . However, the f r a c t i o n s E j and E2 may themselves vary between s p e c i e s . In a d d i t i o n , there has been some overlap between those species observed to produce e i t h e r EOP I or g l u m i t o c i n , and those found to produce the mixture of Ej_ and E 2 . Table I I I summarizes the d i s t r i b u t -ion and d i s t i n g u i s h i n g c h a r a c t e r i s t i c s of the d i f f e r e n t e l a s -mobranch o x y t o c i c p r i n c i p l e s . It i s c l e a r that f u r t h e r s t u d i e s are g r e a t l y needed. I I . THE STATEMENT OF THE PROBLEM The elasmobranch neurohypophysial p r i n c i p l e s , E-[_ , E 2 , EOP I, and EOP I I , had a l r e a d y been r e p o r t e d at the beginning of t h i s study; g l u m i t o c i n was analyzed s h o r t l y a f t e r t h i s work began, and t r a c e s of an a r g i n i n e v a s o t o c i n -l i k e p r i n c i p l e were observed i n s e v e r a l s p e c i e s . The v a r i e t y of neurohypophysial p r i n c i p l e s among the elasmo-branchs appeared gr e a t e r than that which occurs i n any other v e r t e b r a t e c l a s s , and t h e i r exact number and nature was u n c e r t a i n . In the work r e p o r t e d here an e f f o r t was made towards r e s o l v i n g t h i s c o n f u s i o n , both by pharmacological s t u d i e s on crude e x t r a c t s and by the p r e p a r a t i o n of p u r i f i e d p r i n c i p l e s which could be subjected to more r i g o r o u s p h a r m a c o l o g i c a l , chromatographic, and chemical a n a l y s i s . Two species were s t u d i e d : the skate, Raia r h i n a (Jordan and G i l b e r t , 1880) and the d o g f i s h , Squalus acanthias L. ( P a c i f i c v a r i e t y ) = Squalus s u c k l e y i (Gerard, 185*0. These rep r e s e n t e d the two extreme types of elasmobranch p i t u i t a r y s t r u c t u r e ; skates had been found to c o n t a i n the d i f f u s e form of the pars nervosa ( M e u r l i n g , 1967a), and the d o g f i s h showed the d i s c r e t e , and perhaps p r i m i t i v e , form of t h i s p i t u i t a r y r e g i o n ( M e u r l i n g , 1962). Chapter 2 MATERIALS AND METHODS A. C o l l e c t i o n and Storage of the Glands The p i t u i t a r y neurointermediate lobes from two species of elasmobranch, Raia r h i n a (the longnose skate) and Squalus a c a n t h i a s , P a c i f i c v a r i e t y , (the spiny d o g f i s h ) , were obtained by t r a w l f i s h i n g o f f the coast of B r i t i s h Columbia and Washington S t a t e , between 1°66 and 1968. The l o c a t i o n s and dates of the v a r i o u s catches are l i s t e d i n Appendix I. The f i s h were d i s s e c t e d i n l i v i n g c o n d i t i o n on board s h i p . The w a l l s and r o o f of the b r a i n c a s e were removed to r e v e a l the b r a i n . The o p t i c nerves, and the smaller nerves and blood v e s s e l s which attached to the b r a i n , were severed. The connection between the a n t e r i o r lobe of the p i t u i t a r y and the v e n t r a l l o b e , which i s embedded i n the f l o o r of the b r a i n c a s e , was cut. The b r a i n was then p u l l e d back from i t s a n t e r i o r end, and t h e . p i t u i t a r y gland on i t s v e n t r a l surface was exposed. The a n t e r i o r lobe was peeled away, and the neurointermediate lobe was cut f r e e from the u n d e r l y i n g saccus v a s c u l o s u s , and removed. Glands obtained i n t h i s way were t r e a t e d by one of two methods: ko I l l 1. neurointermediate lobes d r i e d i n acetone The lobes were placed i n 25 ml gla s s c o l l e c t i n g b o t t l e s c o n t a i n i n g acetone which had p r e v i o u s l y been d r i e d over CaClg and f i l t e r e d through Whatman #1 paper. U s u a l l y the glands of a p a r t i c u l a r species taken i n one t r a w l were p l a c e d i n the same c o l l e c t i n g b o t t l e . Twenty minutes a f t e r d i s s e c t i o n the acetone was poured o f f and r e p l a c e d with f r e s h acetone. This was repeated at h a l f hour i n t e r v a l s f o r the next two hours, and again a f t e r twenty-four and f o r t y -eight hours. The glands were then d r i e d on an agate mortar over gentle heat (32°C) to remove the l a s t t r a c e s of acetone. For long term storage i n the c o l d (U°C), the glands were e i t h e r kept i n a vacuum d e s s i c a t o r over P^ O,-, or were sealed i n t o g l a s s tubes. 2. L y o p h i l y z e d neurointermediate lobes T i s s u e s c o l l e c t e d during 1968 were l y o p h i l y z e d . F r e s h l y d i s s e c t e d glands were p l a c e d i n 5 ml p o l y e t h y l e n e screw cap v i a l s and fr o z e n on dry i c e . The f r o z e n neuro-intermediate lobes were p l a c e d i n a V i r t i s l y o p h i l y z e r , evacuated to 0.01 mm Hg pressure. A f t e r twenty-four hours the t i s s u e s were removed and s t o r e d at k°C, e i t h e r i n a d e s s i c a t o r over P „ 0 r or i n sealed g l a s s tubes. 2 5 The method by which each sample of glands was t r e a t e d i s l i s t e d i n Appendix I. k2 B. E x t r a c t i o n of the T i s s u e s and Storage of the E x t r a c t The d r i e d t i s s u e s were u s u a l l y e x t r a c t e d at a con-c e n t r a t i o n of ho mg dry t i s s u e / m l i n an aqueous 0 . 2 5 % s o l u t i o n of a c e t i c a c i d . Samples #2 and #3 of Squalus  a c a n t i a s , and samples #1 to #12 of Rai a r h i n a (Appendix I) were e x t r a c t e d i n a 0 . 2 5 % s o l u t i o n of a c e t i c a c i d which contained 0 . 9 % NaCl. Before e x t r a c t i o n , the t i s s u e s were ground f o r approx-imately f i v e minutes i n the a p p r o p r i a t e volume of 0 . 2 5 % a c e t i c a c i d , or i n 0 . 2 5 % a c e t i c a c i d with 0 . 9 % NaCl. This process was c a r r i e d out using a 10 or 50 ml g l a s s e x t r a c t o r (Thomas) f i t t e d with a t e f l o n g r i n d e r (Thomas) and d r i v e n hy an e l e c t r i c motor ( F u l t o r k ) . Then the e x t r a c t o r tube was plugged with cotton and p l a c e d i n a b o i l i n g water bath f o r three minutes. The tube was allowed to c o o l , and the e x t r a c t was f i l t e r e d through Whatman #1 paper. I f l a r g e volumes of e x t r a c t were i n v o l v e d , f i l t r a t i o n was speeded with a Buchner f u n n e l . The f i l t e r e d e x t r a c t s were s t o r e d f o r short periods of time i n screw cap v i a l s i n the c o l d (U°C), without appar-ent l o s s of a c t i v i t y . However e x t r a c t s which were s t o r e d f o r p e r i o d s of s e v e r a l weeks or more were f r o z e n immediately, and kept at - l 8 ° C . C. Methods of B i o l o g i c a l Assay A number of methods of assay, based on the d i f f e r e n t b i o l o g i c a l a c t i v i t i e s of neurohypophysial p e p t i d e s , were h3 used to assess the p o t e n c i e s of the elasmobranch p r i n c i p l e s . These are o u t l i n e d below:-1. Rat Uterus Assay This assay was performed on the i s o l a t e d r a t uterus as o u t l i n e d by Holton (l9*+8) and m o d i f i e d by Munsick ( i 9 6 0 ) . Female Wistar a l b i n o r a t s , weighing 180 to 220 gm and i n n a t u r a l estrous c o n d i t i o n were used. The r a t was stunned by a blow on the head and the blood v e s s e l s of the neck were severed. The u t e r u s , o v a r i e s , and vagina were removed and p l a c e d i n b a t h i n g s o l u t i o n (composition given i n Appendix I I I ) . The two u t e r i n e horns were separated. One of the horns was attached to a muscle hook and supported v e r t i c a l l y , ovary upwards, i n a 5 ml c y l i n d r i c a l muscle bath f i l l e d with bathing s o l u t i o n . The ovary was attached by a s i l k t hread to the p o i n t e r of a smoked drum kymograph. The t i s s u e was aerated with 5% CO^ i n a i r or oxygen throughout the assay. The muscle bath was connected at i t s base to a two l i t e r r e s e r v o i r of bathing s o l u t i o n , which was a l s o aerated with 5% COg i n a i r or oxygen, so that f r e s h s o l u t i o n could be f l u s h e d through the muscle bath between each dose of peptide. The muscle bath and r e s e r v o i r were contained i n a 21 l i t e r water bath maintained at 32°C by a B r o n w i l l C i r c u l a t i n g Motor ( W i l l Corp.). Assays were performed against s y n t h e t i c standards: o x y t o c i n (Syntocinon), g l u m i t o c i n , or a r g i n i n e v a s o t o c i n 1+1+ (see Appendix I I ) , which were f r e s h l y d i l u t e d with water f o r each assay. Unknowns were d i l u t e d with water and n e u t r a l -i z e d with aqueous 2 N NaOH or, l a t e r , with s o l i d NaHCO^. The standards and unknowns were always given i n volumes of 0.20 ml or l e s s . The doses of unknown and standard were d e l i v e r e d i n t o the muscle bath at f i v e minute i n t e r v a l s , f o l l o w i n g the "four p o i n t " s t a t i s t i c a l procedure of Holton (1948). The potency and the f i d u c i a l l i m i t s of the potency, expressed at the 95% confidence l e v e l , were found by the method of Holton (1948). L i m i t s were c a l c u l a t e d only i f an assay of three or more groups of four responses was obtained. Responses were measured as the height i n mm of the r e c o r d of the u t e r i n e c o n t r a c t i o n . 2. Rabbit M i l k - E j e c t i o n Assay The e f f e c t of the unknown in causing e j e c t i o n of milk from a l a c t a t i n g r a b b i t was measured by t h i s assay. In general the method f o l l o w e d was that of van Dyke e_t a l . (1955). The r a b b i t was removed from her young the evening before the assay. An hour before the assay she was given an i n t r a p e r i t o n e a l i n j e c t i o n of 0.65 ml/kg body weight of " D i a l " a n a e s t h e t i c (composition given i n Appendix I I I ) . When the animal was unconscious, the marginal v e i n of one of i t s ears was cannulated with a 22 gauge t u b e r c u l i n h5 needle attached to a 15 cm l e n g t h of PE 50 p o l y e t h y l e n e tubing (Clay Adams); the whole cannula was f i l l e d with an aqueous 0.9% s o l u t i o n of NaCl. The t i p of a t e a t was r e -moved. One of the exposed mammary ducts was cannulated with a b l u n t 20 gauge t u b e r c u l i n needle connected by PE 60 p o l y -ethylene t u b i n g to a venous transducer (Statham P23BB). The cannula, t r a n s d u c e r , and tu b i n g contained a 1% aqueous s o l u t i o n of sodium c i t r a t e . The assays were performed against s y n t h e t i c o x y t o c i n (Syntocinon) or s y n t h e t i c g l u m i t o c i n (see Appendix II) which were f r e s h l y d i l u t e d with aqueous 0.9% NaCl . s o l u t i o n f o r each assay. The unknowns were d i l u t e d with 0.9%> NaCl s o l u -t i o n and n e u t r a l i z e d with aqueous 2 N NaOH or s o l i d NaHCO^ before assay. The standards and unknowns were i n j e c t e d i n t o the ear cannula i n volumes of up to 0.20 ml, and each dose was f o l l o w e d immediately by a 0.20 ml wash of 0.9% NaCl s o l u t i o n . The doses were given at i n t e r v a l s of s i x to ten minutes. The "four p o i n t " s t a t i s t i c a l procedure of Holton (1948) was f o l l o w e d . A f t e r each i n j e c t i o n the r i s e i n intramammary pressure was recorded on a Beckman Type RS Dynograph (Beckman Instruments). Responses were measured as the height of the r e c o r d i n mm above the base l i n e . 3. Frog Bladder Assay The e f f e c t of the unknown i n causing an i n c r e a s e i n the passage of water across the i s o l a t e d f r o g bladder w a l l h6 was measured. The bladder i s p a r t i c u l a r l y s e n s i t i v e to a r g i n i n e v a s o t o c i n , and the assay i s a v a l u a b l e t o o l f o r d e t e c t i n g the presence of t h i s p e p t i d e . The method used was that of Sawyer (i960; and p e r s o n a l communication to Dr. A. M. P e r k s ) . Male b u l l f r o g s (Rana catesbiana) were stunned by a blow on the head. The u r i n a r y bladder was d i s s e c t e d f r e e and p l a c e d i n Ringer's s o l u t i o n (composition given i n Appendix I I I ) . The two lobes of the bladder were cut apart. The neck of each h a l f - b l a d d e r was t i e d with a s i l k thread around the f l a r e d end of a hollow g l a s s cannula ( l e n g t h 9.h cm; diameter 0.9 cm) and the bladder was f i l l e d with 5 ml of d i s t i l l e d water. Each bladder and cannula was suspended by means of the cannula's s i d e arms i n a t i s s u e bath (11 cm high and 2.7 cm i n diameter) which contained 25 ml of Ringer's s o l u t i o n . The bladders were aerated with compres-sed a i r throughout the assay. Assays were performed at room temperature. The bladders were weighed at 15 m i n u t e . i n t e r v a l s u n t i l a constant weight l o s s occurred over three s u c c e s s i v e p e r i o d s . A dose of standard or unknown was added to the outer bath and the bladder was weighed a f t e r 15, 30, and 1+5 minutes. I t was then t r a n s f e r r e d to f r e s h Ringer's and allowed to r e t u r n to i t s r e s t i n g r a t e of weight l o s s before another dose of peptide was given. Assays were performed 47 against s y n t h e t i c o x y t o c i n (Syntocinon) or s y n t h e t i c a r g i n i n e v a s o t o c i n (see Appendix I I ) . Syntocinon was used u n d i l u t e d ; a r g i n i n e v a s o t o c i n was f r e s h l y d i l u t e d with water f o r each assay. Unknowns were n e u t r a l i z e d with aqueous 2 N NaOH or s o l i d NaHCO^ and d e l i v e r e d i n t o the Ringer's s o l u t i o n u n d i -l u t e d . Dose volumes were always 0.20 ml or l e s s . Doses were given by the "four p o i n t " s t a t i s t i c a l procedure of Holton (1948). One group of four responses was obtained on each h a l f - b l a d d e r . Responses were measured as the average of the weight l o s s e s i n the second and t h i r d 15 minute i n t e r v a l s a f t e r the dose was given minus the average of the weight l o s s e s i n the two per i o d s before the dose was given. 4. A n t i d i u r e t i c Assay The a b i l i t y to cause an a n t i d i u r e s i s i n the anaesthe-t i z e d r a t was used as a measure of potency. In g e n e r a l , the method fo l l o w e d was that of J e f f e r s e_t_ al_. (1942), as modi-f i e d by Dicker (1953). A male Wistar a l b i n o r a t of approximately 250 gm was an a e s t h e t i z e d with 5 ml/100 gm body weight of 12% ethanol i n water, given through a stomach tube (#8 French u r e t h r a l c a t h e t e r ) . Two percent aqueous x y l o c a i n e h y d r o c h l o r i d e ( A s t r a Pharmaceuticals) was i n j e c t e d under the s k i n of the neck, and one of the j u g u l a r veins was cannulated with a 20 cm l e n g t h of PE 10 p o l y e t h y l e n e t u b i n g f i l l e d with 0.9% NaCl s o l u t i o n . A t r a c h e a l cannula of PE 50 p o l y e t h y l e n e t u b i n g was i n s e r t e d . X y l o c a i n e h y d r o c h l o r i d e was i n j e c t e d under 1+8 the s k i n above the u r i n a r y bladder and the bladder was can-n u l a t e d with a 15 cm l e n g t h of PE 200 p o l y e t h y l e n e t u b i n g . The penis was t i e d o f f . The r a t was brought to 108% of i t s o r i g i n a l weight with an aqueous s o l u t i o n of 1.5% ethanol and 0.05% NaCl.,. i, given by stomach tube. Throughout the assay, 2 ml of t h i s s o l u t i o n were given as each 2 ml of u r i n e were produced. A f t e r the r a t had come to a constant r a t e of u r i n e flow, i n j e c t i o n s of standard and unknown were begun. Assays were performed against a mixture of a r g i n i n e and l y s i n e v a s o p r e s s i n ( P i t r e s s i n ) or against the s y n t h e t i c p e p t i d e s , a r g i n i n e v a s o t o c i n and g l u m i t o c i n (see Appendix I I ) . These were f r e s h l y d i l u t e d with aqueous 0.9% NaCl s o l u t i o n f o r each assay. Unknowns were d i l u t e d with 0.9% NaCl s o l u t i o n and n e u t r a l i z e d with aqueous 2 N NaOH or s o l i d NaHCO^. The standards and unknowns were i n j e c t e d i n t o the j u g u l a r cannula i n volumes of 0.20 ml or l e s s , and each dose was f o l l o w e d immediately by a wash of 0.20 ml of aqueous 0.9% NaCl s o l u t i o n . The "four p o i n t " procedure of Holton (1948) was f o l l o w e d . The drops of u r i n e were d i r e c t e d over a drop r e c o r d -ing u n i t (Palmer). The arm of an attached timer (Palmer) moved upwards on a kymograph drum at a constant speed between the drops and f e l l whenever a drop was produced. Thus the r e c o r d i n g gave a d i r e c t p i c t u r e of the a n t i d i u r e t i c e f f e c t of an i n j e c t i o n . The responses were q u a n t i t a t e d by the h9 f o l l o w i n g measurements:-m, ,.,. .. h e i g h t o f r e c o r d d u r i n g r e s p o n s e The a n t i d i u r e t i c r e s p o n s e = -—. , ^  =— ; : — f—. h e i g h t o f r e c o r d d u r i n g r e s t i n g f l o w Where: l ) t h e h e i g h t d u r i n g r e s t i n g f l o w = t h e a v e r a g e h e i g h t i n mm o f t h e t r a c i n g s made i n t h e t h r e e m i n u t e s i m m e d i a t e l y p r e c e e d i n g t h e i n j e c t i o n ; and 2) t h e h e i g h t d u r i n g t h e r e s p o n s e = t h e a v e r a g e h e i g h t i n mm o f t h e 20 t o hO t r a c i n g s w h i c h were c e n t e r e d a r o u n d t h e peak o f t h e r e s p o n s e . The number o f t r a c i n g s measured f o r each r e s p o n s e w i t h i n a group o f f o u r was c o n s t a n t ; however t h e number v a r i e d between a s s a y s i n a c c o r d a n c e w i t h t h e c h a r a c t e r i s t i c s o f e a c h p r e p a r a t i o n . 5• A c t i v i t y R a t i o s , Used f o r t h e C o m p a r i s o n o f  D i f f e r e n t A s s a y Methods The d i f f e r e n c e i n p o t e n c y , o r a c t i v i t y , f o u n d f o r an unknown by two d i f f e r e n t methods o f a s s a y was e x p r e s s e d by t h e f o l l o w i n g s e r i e s o f r a t i o s : ^ o r p _ a c t i v i t y on t h e r a t u t e r u s , M g + + p r e s e n t Mg' Mg ~ ++ a c t i v i t y on t h e r a t u t e r u s , no Mg 50 a c t i v i t y on the m i l k - e j e c t i o n assay + + a c t i v i t y on the r a t u t e r u s , no Mg a c t i v i t y on the a n t i d i u r e t i c assay ++ a c t i v i t y on the r a t u t e r u s , no Mg a c t i v i t y on the f r o g bladder assay ++ a c t i v i t y on the r a t u t e r u s , no Mg Where: "R" r e f e r s to the r a t i o s of assays performed against o x y t o c i n (Syntocinon) and vaso-p r e s s i n ( P i t r e s s i n ) standards, as above. and "D" r e f e r s to the r a t i o s of assays performed against other p e p t i d e s , as i n d i c a t e d i n the a p p r o p r i a t e t e x t . The f i d u c i a l l i m i t s of these r a t i o s , expressed at the 95% confidence l e v e l , were found by the f o l l o w i n g formula, s u p p l i e d by B u t l e r , s t a t i s t i c s c o n s u l t a n t to the N u f f i e l d I n s t i t u t e f o r Medical Research, Oxford U n i v e r s i t y ( p ersonal communication to Perks). Where: s u b s c r i p t s 1 and 2 r e f e r to assays 1 and 2 M = l o g R, and R = r a t i o of the dose of unknown to standard S M= standard e r r o r of M, c a l c u l a t e d by the method of Holton (19^8) L i m i t s of M - M = M - Mg 51 t = Student's t , corresponding at the 5% l e v e l of s i g n i f i c a n c e to F degrees of freedom, where F i s c a l c u l a t e d by the method of Brownlee (1965): 2 F = + s M 2 M 2 -M l s M 2 + M 2 n l n 2 where: F = combined degrees of freedom f o r the two as says and n = degrees of freedom i n each assay, c a l c u l a t e d by Holton (19U8 ) . Since the a n t i l o g s of the l i m i t s of M^ - M^ = l i m i t s of R^/Rg, and since = P^ and Rg k2 = P 2 where P = potency found f o r the unknown i n each assay , , , dose of standard ( d i l u t i o n of unknown) and k = constant = - 5 — : -volume of unknown then, ( l i m i t s of R /R ) (k-^k ) = l i m i t s of P-j/Pg ' D . N o n - B i o l o g i c a l Measurements 1. Measurement of P r o t e i n or Peptide Content The p u r i t y of the neurohypophysial peptide s o l u t i o n s 52 was estimated by the r a t i o of t h e i r b i o l o g i c a l a c t i v i t y to t h e i r peptide content ( " s p e c i f i c a c t i v i t y " ) . P r o t e i n or peptid e content was measured by the method of Lowry et_ a l . (1951). The unknowns were measured against bovine serum albumin (Armour). A s e r i e s of aqueous s o l u t i o n s of the standard, ranging from 25 ug/ml to 500 yg/ml, were prepared f r e s h l y from a stock s o l u t i o n which was made at 0.05 gm/ml. Unknowns were d i l u t e d with water to the a p p r o p r i a t e concen-t r a t i o n . One ml of a l k a l i n e copper s o l u t i o n (see Appendix IV) was added to 0.20 ml of each standard and unknown, and to a water blank. The s o l u t i o n s were mixed w e l l and allowed to rea c t f o r ten minutes at room temperature. Then 0.10 ml phosphomolybdic-phosphotungstic a c i d reagent (see Appendix IV) was added to each and mixed immediately on a Genie-Vortex S t i r r e r ( F i s h e r S c i e n t i f i c ) . The samples were l e f t at room temperature f o r one hour, during which time a blue colour developed. The o p t i c a l d e n s i t y of the unknowns and standards was measured at X = 750 my on a Unicam Spectrophotometer SP 600, m o d i f i e d to accommodate small volumes. A standard curve was drawn and the peptide c o n c e n t r a t i o n of the unknowns was read from i t . 53 2. Measurement o f C o n d u c t i v i t y S p e c i f i c c o n d u c t i v i t y m e a s u r e m e n t s were u s e d f o r t h e r a p i d a p p r o x i m a t i o n o f s a l t c o n c e n t r a t i o n s d u r i n g t h e i o n e x c h a n g e c h r o m a t o g r a p h y p r o c e d u r e s . The c o n d u c t i v i t y o f t h e s o l u t i o n s was m e a s u r e d d i r e c t l y on a R a d i o m e t e r C o n d u c t i v i t y M e t e r Type CDM 2d, and was e x p r e s s e d as t h e s p e c i f i c c o n d u c -t i v i t y , mho/cm. 3. I n a c t i v a t i o n w i t h S o dium T h i o g l y c o l l a t e " N e u r o h y p o p h y s i a l p e p t i d e s l o s e t h e i r b i o l o g i c a l a c t i v i t y a f t e r i n c u b a t i o n w i t h s o d i u m t h i o g l y c o l l a t e a t a n e u t r a l pH ( v a n Dyke et_ a_l. , 19^2). Unknowns were t e s t e d by t h e method o f V o g t (1953). One p a r t o f 0.1 N s o d i u m t h i o g l y -c o l l a t e was added t o n i n e p a r t s o f t h e t e s t s o l u t i o n . The m i x t u r e was n e u t r a l i z e d w i t h 2 N NaOH and l e f t a t room t e m p e r a t u r e f o r one h o u r . The e x t e n t o f i n a c t i v a t i o n was m e a s u r e d by a s s a y on t h e r a t u t e r u s . h. P a p e r C h r o m a t o g r a p h y The e l a s m o b r a n c h p r i n c i p l e s were s u b j e c t e d t o p a p e r c h r o m a t o g r a p h y f o r p a r t i a l i s o l a t i o n and p u r i f i c a t i o n , and f o r t h e c o m p a r i s o n o f t h e i r c h r o m a t o g r a p h i c b e h a v i o r w i t h t h a t o f known p e p t i d e s . D e s c e n d i n g c h r o m a t o g r a m s were r u n on Whatman 3MM p a p e r , 52 cm by 22 cm, as d e s c r i b e d by P e r k s (1966). The s a m p l e s were a p p l i e d 2 i n c h e s b e l o w t h e a n t i - s i p h o n r o d . Two o r t h r e e o r i g i n s , e a c h 2.5 cm l o n g , were s p a c e d a c r o s s 54 the chromatogram between outer margins of 4.0 cm. Samples ranging from 20 to 1,100 mU r a t uterus a c t i v i t y and from 0.02 ml to 0.67 ml i n volume were a p p l i e d to a given o r i g i n . During most experiments, one or two standards of known chromatographic behavior - such as s y n t h e t i c o x y t o c i n (Syntocinon), a r g i n i n e / l y s i n e v a s o p r e s s i n ( P i t r e s s i n ) , or s y n t h e t i c a r g i n i n e v a s o t o c i n - were run next to the unknown. The s o l v e n t system which was used was n - b u t a n o l : a c e t i c a c i d : water = 4:1:5 ( H e l l e r and P i c k e r i n g , 1961). The lower s o l -vent phase was placed at the bottom of the chromatography tank. E q u i l i b r a t i o n time was two hours; at midpoint the chromatogram was p o s i t i o n e d i n the tank. The upper s o l v e n t phase was run i n t o the sol v e n t trough and the chromatogram was developed f o r twelve hours at room temperature (approx-imately 22°C). A f t e r development, the paper was d r i e d under a stream of c o o l a i r . Below each o r i g i n a l o n g i t u d i n a l s t r i p was cut; i t ran from the o r i g i n to the solvent f r o n t , and extended 0.5 cm on each si d e of the o r i g i n . Each s t r i p was cut i n t o ten equal s e c t i o n s . Each s e c t i o n was pl a c e d i n 1 ml of an aqueous 0.25% s o l u t i o n of a c e t i c a c i d or i n 1 ml of 0.25% a c e t i c a c i d c o n t a i n i n g 0.9% NaCl, and l e f t i n the c o l d f o r one hour. The f l u i d content was expressed manually from each s e c t i o n and mixed with that which remained i n the beaker. The eluates were n e u t r a l i z e d with aqueous 2 N NaOH or s o l i d NaHCO , and assayed on the a p p r o p r i a t e p r e p a r a t i o n 55 to l o c a t e the a c t i v e p e p t i d e s . Recovery of the a c t i v e p r i n -c i p l e s from the paper u s u a l l y approximated 33%. On one o c c a s i o n , one l a r g e sample (3.6k IU r a t uterus a c t i v i t y , no Mg + + present) of Squalus e x t r a c t was subjected to paper chromatography i n the above solvent system, as a f i n a l step a f t e r , i t s p u r i f i c a t i o n by g e l f i l t r a t i o n and i o n exchange chromatography. In t h i s case the o r i g i n was 3 cm across and the width of the e l u t e d s t r i p was 5 cm. It was d i v i d e d i n t o twenty s e c t i o n s , and these were e l u t e d three times i n t o 2 ml of 0.25% a c e t i c a c i d , i n order to recover as much a c t i v i t y as p o s s i b l e . 5• I s o e l e c t r i c Focusing Two p u r i f i e d elasmobranch p r e p a r a t i o n s were subjec-ted to i s o e l e c t r i c f o c u s i n g (Svensson, 196l; 1962a; 1962b; Vesterberg and Svensson, 1966). By t h i s procedure substances whose i s o e l e c t r i c p o i n t s d i f f e r by as l i t t l e as 0.02 pH u n i t s can be separated. An LKB 8101 e l e c t r o f o c u s i n g column (LKB P r o d u k t e r ) , of 110 ml c a p a c i t y , was used, a c c o r d i n g to the i n s t r u c t i o n s of the manufacturer. A s e r i e s of ampholyte-suerose f r a c t i o n s of i n c r e a s i n g d e n s i t y was prepared (see Appendix IV). The peptide sample was i n c o r p o r a t e d i n t o one of the middle f r a c t i o n s ; sample volumes were approximately 1 ml. The ampholyte-sucrose f r a c t i o n s were poured i n t o the e l e c t r o -f o c u s i n g column i n order of d e c r e a s i n g d e n s i t y ; they were 56 preceeded i n t o the column by a cathode s o l u t i o n (see Appendix IV) and followed by an anode s o l u t i o n (see Appendix IV). Runs were made at 300 v o l t s f o r 4 8 hours (ampholyte pH range 3-10) or at 800 v o l t s f o r 25 hours (ampholyte pH range 7-9). The column was drained at a r a t e of 1.7 ml/min. F r a c t i o n s of 0.75 ml or 1.0 ml were c o l l e c t e d i n an LKB F r a c t i o n C o l -l e c t o r (LKB Produkter) and assayed on the r a t uterus i n order to l o c a t e the p o s i t i o n of the unknown peptide i n the newly e s t a b l i s h e d pH g r a d i e n t . E. P u r i f i c a t i o n Procedures 1. Gel F i l t r a t i o n The f i r s t step i n the p u r i f i c a t i o n of the neuro-h y p o p h y s i a l p eptides was t h e i r s e p a r a t i o n from the l a r g e p r o t e i n molecules present i n the crude e x t r a c t s . To achieve t h i s , the e x t r a c t s were f i l t e r e d through a column of Sephadex G-15 (Pharmacia Fine Chemicals), a g e l which separates molecules on the b a s i s of s i z e . The g e l was prepared i n 0.2 M a c e t i c a c i d , which was a l s o used as the eluant. 150 gm of Sephadex G-15 (Pharmacia), Lot T08507, was added to 2 l i t e r s of 0.2 M a c e t i c a c i d . The mixture was s t i r r e d f o r 5 minutes and l e f t to s e t t l e f o r one hour. The supernatant, c o n t a i n i n g the " f i n e s " , was decanted. This was repeated s i x times, i n c l u d i n g one overnight s t i r r i n g p e r i o d , u s i n g 2 l i t e r s of 0.2 M a c e t i c a c i d each time. A l a r g e f u n n e l was f i t t e d i n t o the top of a g l a s s chromatography 57 column (Kontes; height 100- cm; inner diameter 2.k cm) and the column and part of the fu n n e l were f i l l e d with 0.2 M a c e t i c a c i d . A l i q u o t s of the swollen g e l were added to the funn e l and allowed to s e t t l e s l o w l y , with continuous s t i r -r i n g , i n t o the column. A f t e r the g e l had "built up to a height of 3 cm the stopcock was opened s l i g h t l y , and l e f t open f o r the remainder of the b u i l d i n g p e r i o d . The column was b u i l t to a height of 90 cm, and a c i r c l e of Whatman #1 paper was pl a c e d over the g e l s u r f a c e . T h i r t e e n samples of crude e x t r a c t were passed through the column at room temperature (approximately 22°C). Between uses, the column was washed with 0.2 M a c e t i c a c i d and stored i n the c o l d (U°C). In order to o b t a i n b e t t e r s e p a r a t i o n of the neurohypophysial peptides from the l a r g e r p r o t e i n s , the s i z e of the samples a p p l i e d to the column was r e s t r i c t e d to 10 ml. However, i n l a t e r experiments the sample volumes were i n c r e a s e d to as much as 35 ml i n order to reduce the d i l u t i o n of the pep t i d e s during t h e i r passage through the g e l . Before the sample was a p p l i e d , the a c e t i c a c i d i n the column was dra i n e d down to the sur f a c e of the g e l . The e x t r a c t was c a r e f u l l y added dropwise onto the top of the g e l , and the f l u i d was allowed to run slowly i n t o the s u r f a c e . 5 ml of 0.2 M a c e t i c a c i d were then a p p l i e d i n the same way and allowed to run i n t o the g e l . The column was f i l l e d to the top with 0.2 M a c e t i c a c i d and connected through p o l y e t h y l e n e t u b i n g to a r e s e r v o i r of 0.2 M a c e t i c a c i d . The 58 r a t e of flow of the eluant through the column was set at 0.3 ml/min. The eluate was l e d through PE 260 p o l y e t h y l e n e t u b i n g up to a f r a c t i o n c o l l e c t o r l o c a t e d on a l e v e l with the top of the column. The s i z e of the f r a c t i o n s v a r i e d from 2 to 5 ml during d i f f e r e n t runs. The f r a c t i o n s were t e s t e d , as r e q u i r e d , f o r r a t uterus a c t i v i t y , Lowry peptide c o n c e n t r a t i o n , and f o r c o n d u c t i v i t y . 2. F i l t r a t i o n Through Ion R e t a r d a t i o n Resin A f t e r g e l f i l t r a t i o n , s e v e r a l of the peptide s o l u -t i o n s were passed through an ion r e t a r d a t i o n r e s i n which removed some of t h e i r s a l t content and so f a c i l i t a t e d the a d s o r p t i o n of the peptides onto subsequent Sephadex c a t i o n exchangers. Biorad Ion R e t a r d a t i o n Resin AG11A8, Lot 5867, was used. Burettes (volume 25 ml; inner diameter 0.9 cm) were used as columns. For each column, 20 gm of r e s i n was taken up i n 50 ml water and poured i n t o the column to a height of hO cm, with the ;stopc.oicrfe. s l i g h t l y open. A wash of 200 ml of 0.2 M a c e t i c a c i d was run through the r e s i n . This was f o l l o w e d by s u f f i c i e n t water (approximately 1 l i t e r ) to reduce the s p e c i f i c c o n d u c t i v i t y of the e l u a t e to 0.05 mmho/ cm. The column was s t o r e d i n the c o l d . The sample was passed through the r e s i n at room temperature, f o l l o w e d by water u n t i l no more b i o l o g i c a l a c t i v i t y c ould be detected i n the e l u a t e . Flow r a t e was 59 1.2 ml/min. The recovery of a c t i v i t y was complete. The used r e s i n was d i s c a r d e d . 3. F l a s h E vaporation The a c e t i c a c i d content of the el u a t e s of the Sephadex G-15 and the i o n r e t a r d a t i o n columns i n t e r f e r e d with the a d s o r p t i o n of the peptides onto the subsequent i o n exchange g e l s . Therefore i t was p a r t i a l l y removed from the s o l u t i o n s by f l a s h e vaporation. A h o r i z o n t a l f l a s h evaporator (Bodine E l e c t r i c ) of 500 ml c a p a c i t y was used. The f l a s k c o n t a i n i n g the s o l u t i o n to be evaporated was r o t a t e d i n a water bath maintained at 2T°C. The condensor was at f i r s t bathed i n a stream of c o l d water but i n l a t e r experiments a bath of methanol and dry ic e was used. The condensor was connected to e i t h e r a water a s p i r a t o r o r, through a t r a p , to a Duoseal Vacuum pump (Welch S c i e n t i f i c ) . When the pump was used, a pressure of 0.1 mm Hg was maintained i n the system. The s o l u t i o n was evaporated j u s t to the po i n t of dryness and the r e s i d u e , c o n t a i n i n g the neurohypophysial p e p t i d e s , was taken up i n d i s t i l l e d water. Between 66% and 100% of the o x y t o c i c a c t i v i t y was recovered. h. Ion Exchange Chromatography A f t e r g e l f i l t r a t i o n the peptides were f u r t h e r p u r i -f i e d by chromatography on columns of Sephadex ion exchangers (Pharmacia). Three gels were used. They were die t h y l a m i n o -6o e t h y l (DEAE) Sephadex, carboxymethy1 (CM) Sephadex, and s u l f o e t h y l (SE) Sephadex. The neurohypophysial peptides were not r e t a i n e d "by the p o s i t i v e l y charged groups of the DEAE Sephadex. Nevertheless some necessary p u r i f i c a t i o n was achieved because of the a d s o r p t i o n onto the g e l of nega-t i v e l y charged i m p u r i t i e s , some of which have been shown to i n t e r f e r e with l a t e r procedures (Perks and Sawyer, 1965). However the peptides were adsorbed and concentrated by the n e g a t i v e l y charged CM and SE exchangers. Each exchanger w i l l be d i s c u s s e d s e p a r a t e l y below. a. D i e t h y l a m i n o e t h y l (DEAE) Sephadex DEAE Sephadex, A-25 coarse, Lot T04751, was used throughout. The g e l was prepared according to the manufac-t u r e r ' s i n s t r u c t i o n s : 15 gm of dry g e l was swollen i n 200 ml of d i s t i l l e d water f o r one hour with s t i r r i n g , allowed to s e t t l e , and the water and " f i n e s " were poured o f f . This was repeated twice. The g e l was t r a n s f e r r e d to a Buchner f u n n e l and washed under s u c t i o n with: 1) 100 ml of 0.5 N HC1, f o l l o w e d by 1 l i t e r d i s t i l l e d water 2) 100 ml of 0.5 N NaOH, fol l o w e d by 1 l i t e r d i s t i l l e d water 3) 100 ml of 0.5 N CH 3C00H, fo l l o w e d by 1 l i t e r d i s t i l l e d wat er k) 100 ml of 0.2 M NH^acetate (pH 7.0). The exchanger was t r a n s f e r r e d to a beaker and succes-s i v e l y e q u i l i b r a t e d with 0.2 M, 0.02 M, and 0.002 M s o l u t i o n s of NH, acetate (pH 7.0). The NH, acetate b u f f e r s had been 61 prepared "by t i t r a t i o n of a c e t i c a c i d s o l u t i o n s at the d e s i r e d m o l a r i t y with concentrated NH^OH u n t i l the pH reached 7.0 (Radiometer, pH meter type PHM 22). Burettes (volume 25 ml; inner diameter 0.9 cm) were used as columns. These were set up and "built as d e s c r i b e d f o r the Sephadex G-15 column, except the stopcock was c l o s e d throughout the b u i l d i n g p e r i o d . The columns were b u i l t i n 0.002 M NH^acetatec (pH 7.0) to a height of 35 cm. Then 0.002 M NH^acetatee (pH 7.0, s p e c i f i c c o n d u c t i v i t y 0.21 mmho/cm) was run through the gel u n t i l the s p e c i f i c conduc-t i v i t y of the elua t e measured 0.21 mmho/cm. The columns were stored at k°C. A few samples were run through the columns i n the c o l d (lt°C), but most runs were made at room temperature (approximately 22°C). The sample was adjus t e d to a s p e c i f i c c o n d u c t i v i t y of 0.21 mmho/cm and pH of 7.0 by d i l u t i o n with d i s t i l l e d water and the dropwise a d d i t i o n of M^OH. A p r e l i m i n a r y wash of 50 ml of 0.002 M IH^acetate b u f f e r was run through the column. The sample was then run i n t o the g e l , f o l l o w e d by another 20 ml of the b u f f e r . The flow r a t e through the column was set between 0.5 and 1.0 ml/min. Between 80% and 100% of the b i o l o g i c a l a c t i v i t y was recovered i n the e l u a t e . The used exchanger was d i s c a r d e d . b. Carboxymethyl (CM) Sephadex Two d i f f e r e n t l o t s of CM Sephadex exchanger were 62 used: C-25 medium, Lot TO607I, and C-25, bead type, Lot 8852. Both were prepared i n the same way: 15 gm of dry g e l was swollen i n d i s t i l l e d water as d e s c r i b e d f o r DEAE Sephadex, t r a n s f e r r e d to a Buchner funnel and washed under s u c t i o n with: 1) 100 ml of 0.5 N NaOH, fo l l o w e d by 1 l i t e r d i s t i l l e d water 2) 100 ml of 0.5 N HC1, f o l l o w e d by 1 l i t e r d i s t i l l e d water 3) 100 ml of 0.5 N NH^OH, fol l o w e d by 1 l i t e r d i s t i l l e d water k) 100 ml of 0.2 M NH^acetate (pH 5.0). The exchanger was t r a n s f e r r e d to a beaker and succes-s i v e l y e q u i l i b r a t e d with 0.2 M, 0.02 M, and 0.002 M s o l u t i o n s of NH^acetate (pH 5.0). At l e a s t f i v e changes of the 0.002 M b u f f e r were r e q u i r e d . The NH^acetate b u f f e r s had been pre-pared by adding concentrated NH^OH dropwise and with c o n t i n u -ous s t i r r i n g to s o l u t i o n s of a c e t i c a c i d at the d e s i r e d m o l a r i t y u n t i l the pH reached 5.0 (Radiometer, pH meter type PHM 22). The columns were b u i l t i n 25 ml b u r e t t e s as de s c r i b e d f o r the DEAE Sephadex, except that 0.002 M NH^acetate (pH 5.0) was used. A f t e r a column was b u i l t , 0.002 M NH^acetate (pH 5.0, s p e c i f i c c o n d u c t i v i t y 0.l6 mmho/cm) was run through u n t i l the s p e c i f i c c o n d u c t i v i t y of the elua t e approached 0.l6 mmho/cm. The columns were stored i n the c o l d (4°C). 63 A few runs were made i n the. c o l d (4°C), hut most samples were a p p l i e d to the g e l at room temperature (approx-imately 22°C). The sample, which had j u s t been passed through DEAE Sephadex, was adjusted to a s p e c i f i c conduc-t i v i t y of 0.l6 mmho/cm and pH of 5.0 by d i l u t i o n with d i s t i l -l e d water and the dropwise a d d i t i o n of a c e t i c a c i d . The g e l was washed through with 50 ml of 0.002 M HH^acetate (pH 5.0) and then the sample was a p p l i e d . When the l a s t of the sample s o l u t i o n had passed i n t o the g e l , 1 ml of 0.002 M b u f f e r was added to the top of the g e l and allowed to run i n . This was repeated twice and then the column was f i l l e d to the top with b u f f e r . The p e p t i d e s were e l u t e d by p a s s i n g NH^acetate b u f f e r of i n c r e a s i n g i o n i c s t r e n g t h through the g e l . This was done through a mixing chamber, a 45 ml stoppered g l a s s v i a l on a magnetic s t i r r e r , which was l o c a t e d between the b u f f e r r e s e r v o i r and the top of the column below. I n i t i a l l y the system was f i l l e d with the d i l u t e b u f f e r i n which the column had been b u i l t . This was r e p l a c e d i n the upper r e s e r v o i r by a more concentrated b u f f e r and so the c o n c e n t r a t i o n of b u f f e r e n t e r i n g the column g r a d u a l l y i n c r e a s e d from that of the d i l u t e b u f f e r to that of the more concentrated b u f f e r . The n e u t r a l p e p t i d e s were e l u t e d o f f on t h i s f i r s t g r a d i e n t . Then the r e s e r v o i r was f i l l e d with an even more concentrated b u f f e r , and the b a s i c peptide was e l u t e d along the r e s u l t i n g second g r a d i e n t . 64 The c o n c e n t r a t i o n of "buffer necessary to desorb the n e u t r a l p e p t i d e s d i f f e r e d with each of the two l o t s of CM Sephadex used, and t h i s caused c o n s i d e r a b l e t e c h n i c a l d i f f i -c u l t y i n the e a r l i e r stages of the i n v e s t i g a t i o n . F u r t h e r , a higher c o n c e n t r a t i o n was employed to e l u t e the n e u t r a l peptide of the sakte (Raia r h i n a ) than was used f o r the n e u t r a l f r a c t i o n of the d o g f i s h (Squalus a c a n t h i a s ) . The n e u t r a l f r a c t i o n of Squalus was e l u t e d on a gradient between 0.002 M WH^acetate (pH 5.0, s p e c i f i c c o n d u c t i v i t y 0.l6 mmho/cm) and e i t h e r 0.05 M SfH^acetate (pH 5.0, s p e c i f i c c o n d u c t i v i t y 3.4 mmho/cm), when CM Sephadex Lot T06071 was used, or 0.10 M NH^acetate (pH 5.0, s p e c i f i c c o n d u c t i v i t y 6.2 mmho/cm) i n the case of Lot 8852. The b a s i c f r a c t i o n was e l u t e d from both l o t s of g e l on a second gra d i e n t l e a d -ing up to 0.2 M NH^acetate (pH 7.0, s p e c i f i c c o n d u c t i v i t y 16.4 mmho/cm). The n e u t r a l f r a c t i o n of Raia was e l u t e d from the g e l of Lot 8852 on a gradi e n t between 0.002 M NH^acetate (pH 5.0) and 0.15 M NH^acetate (pH 5-0, s p e c i f i c conduc-t i v i t y 9«2 mmho/cm). The b a s i c f r a c t i o n was e l u t e d on a second g r a d i e n t l e a d i n g up to 0.2 M NH^acetate (pH 7-0). The n e u t r a l f r a c t i o n of Raia was r e - a p p l i e d to CM Sephadex Lot 8852 and e l u t e d on a gradi e n t between 0.022 M NH^acetate (pH 5.0, s p e c i f i c c o n d u c t i v i t y 1.7 mmho/cm) and 0.2 M NH^ace-t a t e (pH 5.0, s p e c i f i c c o n d u c t i v i t y 12.3 mmho/cm). The flow r a t e through the column was l i m i t e d by the r e s i s t a n c e of the g e l ; i n a l l experiments the value was 65 0 . 3 ml/min. The elua t e was c o l l e c t e d i n f r a c t i o n s of between 3.5 and 4.8 ml. The l o c a t i o n of the neurohypophysial pep-t i d e s was determined by r a t uterus and a n t i d i u r e t i c assay. Each column was used only once. c. S u l f o e t h y l (SE) Sephadex One sample of exchanger, Lot T 0 6 7 7 3 , was used through-out. 10 gm of dry g e l was swollen i n d i s t i l l e d water as de s c r i b e d f o r DEAE Sephadex, t r a n s f e r r e d to a Buchner funnel and washed under s u c t i o n with 0 . 5 N s o l u t i o n s of NaOH, H C 1 , and NH^OH, and with 100 ml of 2 M M^formate (pH 2.45). The g e l was t r a n s f e r r e d to a beaker and s u c c e s s i v e l y e q u i l i b r a t e d with 2 M and then with 0 . l 4 M NH^formate (pH 2.45). 2 M and 1 . 5 M NH^formate b u f f e r s were made by adding concentrated NH^OH dropwise and with continuous s t i r r i n g to s o l u t i o n s of 2 M and 1 . 5 M formic a c i d u n t i l the pH reached 2.45. The 0 . l 4 M b u f f e r was prepared from the 2 M b u f f e r as f o l l o w s : A s o l u t i o n of 2 M NH^formate was d i l u t e d with water to a c o n c e n t r a t i o n of 0 . 0 5 M. The pH, which had r i s e n upon d i l u t i o n , was lowered to 2.45 by a d d i t i o n of a measured volume of formic a c i d . The f i n a l formate c o n c e n t r a t i o n of the b u f f e r was c a l c u l a t e d and found to be 0 . l 4 M. The columns were b u i l t i n 0 . l 4 M NH^formate i n 25 ml bu r e t t e s as d e s c r i b e d f o r DEAE Sephadex. A f t e r a column was b u i l t i t was e q u i l i b r a t e d with 0 . l 4 M NH^fo'rmate (pH 2.45, s p e c i f i c c o n d u c t i v i t y 1 . 9 mmho/cm) u n t i l the s p e c i f i c con-66 d u c t i v i t y of the el u a t e had f a l l e n to 1.9 mmho/cm. The columns were stored at h°C. The samples which were a p p l i e d to t h i s gel'con^ t a i n e d only n e u t r a l neurohypophysial p e p t i d e s . A l l were a p p l i e d and e l u t e d at room temperature (approximately 22°C). They were adjusted to s p e c i f i c c o n d u c t i v i t y of 1.9 mmho/cm and pH 2.1+5 "by d i l u t i o n with d i s t i l l e d water and the drop-wise a d d i t i o n of formic a c i d , and run i n t o the g e l i n the manner d e s c r i b e d f o r CM Sephadex. The flow r a t e during l o a d i n g was set at between 0.35 and 0.55 ml/min. The peptides were e l u t e d on an NH^formate concentra-t i o n g r a d i e n t , i n the manner d e s c r i b e d f o r CM Sephadex; the range used was from 0.1k M to 1.5 M b u f f e r (pH 2.U5, s p e c i f i c c o n d u c t i v i t i e s 1.9 and 15.0 mmho/cm, r e s p e c t i v e l y ) . The e l u t i o n flow r a t e was set at between 0.7 and 0.9 ml/min and the s i z e of the f r a c t i o n s c o l l e c t e d was 3.4 ml. The l o c a t i o n of the p r i n c i p l e s i n the el u a t e was detected by the ra t uterus assay. Each column was used only once. 5. L y o p h i l y z a t i o n (Freeze Drying) P a r t i a l e l i m i n a t i o n of the b u f f e r s a l t s from the elu a t e s of the CM and SE exchangers was e f f e c t e d by l y o p h i l y -zing the s o l u t i o n s to dryness on a V i r t i s l y o p h i l y z e r evacuated to 0.01 mm Hg pr e s s u r e . For more complete removal of the ,:NH^.acetate or WH^formate the d r i e d r e s i d u e s were taken up i n water and l y o p h i l y z e d s e v e r a l times. 67 6• H y d r o l y s i s arid Amino A c i d A n a l y s i s The l y o p h i l y z e d samples were taken up i n approximately 3 ml of d i s t i l l e d water and t r a n s f e r r e d to 5 ml Pyrex t e a r -d r y i n g bulbs ( V i r t i s ) . Each was again l y o p h i l y z e d to dry-ness. h.O ml of t r i p l e d i s t i l l e d HC1 was added to the bulb. L i q u i f i e d phenol, 0.Ok ml, was a l s o added to the e a r l y samples to minimize the breakdown of t y r o s i n e (Dixon, p e r s o n a l com-munication) but t h i s was found u n s a t i s f a c t o r y f o r s u l f u r -c o n t a i n i n g compounds and was omitted from the l a t e r samples. Ng gas was bubbled through the sample f o r ten minutes as a b r i s k stream of f i n e bubbles; a f t e r t h i s time the sample was f r o z e n by immersion of the bulb i n l i q u i d N . The bulb was then attached to a Duoseal vacuum pump (Welch S c i e n t i f i c ) o p e r a t i n g at 0.01 mm Hg p r e s s u r e , evacuated f o r 30 minutes, and s e a l e d with a flame while under vacuum. The sealed bulb was p l a c e d i n a hot a i r oven at 107°C and l e f t f o r 18 hours. A f t e r h y d r o l y s i s the contents were t r a n s f e r r e d to a 5 or 10 ml pear shaped f l a s h - e v a p o r a t i o n f l a s k . The sample was p l a c e d i n the f r e e z e r f o r 15 minutes, attached while s t i l l c o l d to a f l a s h evaporator, and taken to dryness over a water bath maintained at 30-35°C. 2 or h ml of r e f r i g e r a t e d water were added to the f l a s k and the evaporation was repeated. The a d d i t i o n of water and subse-quent e v a p o r a t i o n was repeated s e v e r a l times, u n t i l t here was no smell of HC1 i n the sample. The hydrolyzed samples 68 were s t o r e d dry i n the f r e e z e r (-l8°C). The h y d r o l y s a t e s and two c o n t r o l samples were a n a l -yzed f o r amino a c i d c o n t e n t on a Beckman model 120 Amino A c i d A n a l y z e r or on a B i o c a l BC-200 Amino A c i d A n a l y z e r . The r a t u t e r u s a c t i v i t y of the p e p t i d e i n the samples which were s u b s e q u e n t l y h y d r o l y z e d and a n a l y z e d ranged from 600 t o 2880 mU. Chapter 3 RESULTS I. STUDIES OF THE NEUROHYPOPHYSIAL PRINCIPLES OF THE SKATE, RAIA RHINA A. Int ro duct i on The skate, Rai a r h i n a , was s t u d i e d i n order to extend the work on the elasmobranch neurohypophysis to a new P a c i f i c s p e c i e s . T h i s species was of i n t e r e s t because i t belonged to a genus whose v a r i o u s members had already been r e p o r t e d to produce the f u l l range of elasmobranch p r i n c i p l e s - EOP I, EOP I I , E l 9 E 2 , and g l u m i t o c i n (Table I I I , page 33). F u r t h e r , the p r i n c i p l e s produced by the d i f f u s e type of pars nervosa found i n the skates ( M e u r l i n g , 1967a) might form an i n t e r e s t i n g c o n t r a s t with those produced by the d i s c r e t e type of neuro-hypophysis present i n Squalus acanthias , the p r i n c i p a l species s t u d i e d here ( M e u r l i n g , 1962). In a d d i t i o n , Manning's s y n t h e s i s of g l u m i t o c i n (Manning et_ a_l. , 1968) during the course of t h i s work meant that a d i r e c t p h armacological comparison could be made between the skate neurohypophysial p r i n c i p l e and a standard of p o s s i b l y i d e n t i c a l molecular s t r u c t u r e . 6"9> 70 B. Pharmacological and Chromatographic Studies of the  Neurointermediate Lobe E x t r a c t s 1. B i o l o g i c a l A c t i v i t i e s and A c t i v i t y Ratios of  the Crude E x t r a c t F i f t e e n glands (sample 6, see Appendix I) were e x t r a c t e d at 40 mg d r i e d t i s s u e / m l . They were assayed a g a i n s t s y n t h e t i c o x y t o c i n (Syntocinon, Sandoz) f o r r a t uterus and m i l k - e j e c t i o n a c t i v i t y , and against v a s o p r e s s i n ( P i t r e s s i n , Parke Davis) f o r a n t i d i u r e t i c a c t i v i t y . The a c t i v i t y r a t i o s found f o r t h i s species were compared to those of other elasmobranchs. The glands were found to have a r a t uterus a c t i v i t y of 2.0 ± 0.3 mU/mg d r i e d t i s s u e . This l e v e l of a c t i v i t y was low even f o r elasmobranchs (Perks and Dodd, 1963a), although s i m i l a r low l e v e l s occur i n c e r t a i n species of l a r g e sharks (Perks, 1966). The m i l k - e j e c t i o n a c t i v i t y was 8.7 mU/mg and the a n t i d i u r e t i c a c t i v i t y was 0.03 mU/mg. The r a t i o s of the a c t i v i t i e s , ME ( m i l k - e j e c t i o n ) : RU (rat u t e r u s ) : ADH ( a n t i -d i u r e t i c ) , were 4.4:1:0.02. This was i n agreement with values found f o r most other elasmobranchs, i n c l u d i n g two skates, R. c l a v a t a and R. bat i s; the values c h a r a c t e r i z e d the R. r h i n a p r i n c i p l e as the EOP I type, which i s d i s t i n g u i -shed by an R„„ of approximately 3, ra t h e r than as the p r i n c i p l e designated as EOP I I , i n which the R i s approximately 10 (Perks and Dodd, 1963a; Sawyer, 1964; Perks, 1966). 71 2. Studies of the Magnesium P o t e n t i a t i o n of the  E x t r a c t s a. The range of magnesium p o t e n t i a t i o n s i n  twelve crude e x t r a c t s I t was p o s s i b l e that the R. r h i n a e x t r a c t could c o n t a i n a mixture of s e v e r a l n e u t r a l p e p t i d e s , f o r there had been c o n f l i c t i n g r e p o r t s on the nature and number of p r i n c i p l e s which c o n s t i t u t e d the EOP I f r a c t i o n i n s e v e r a l other elasmobranch s p e c i e s . H e l l e r and P i c k e r i n g (1961), H e l l e r and Roy (1965a), and Roy (1969) r e p o r t e d that two o x y t o c i c p r i n c i p l e s could be separated by paper chromatography from e x t r a c t s of Squalus ac anthias (spiny d o g f i s h ) , Carcharinus 1eucas ( b u l l s h a r k ) , and Raia c l a v a t a (thornback r a y ) , three species which gave a pharmacological spectrum of the EOP I type (Perks, 1966). These agents were designated as E^ and E^: E , the slow-running p r i n c i p l e , was f u r t h e r c h a r a c t e r i z e d i n some species by the high degree to which i t s a c t i v i t y on the r a t uterus was p o t e n t i a t e d by magnesium i o n s , w h i l s t i n c o n t r a s t , E^ , the f a s t - r u n n i n g p r i n c i p l e , was only moderately magnesium p o t e n t i a t e d (see Table I I I , page 33). Other i n v e s t i g a t o r s (Perks, 1966; Sawyer, 1967, Swiatkiewicz, 1968) were unable to confirm these r e s u l t s . They found that chromatograms of Squalus and C a r c h a r i n u s , developed i n the same solvent system - n - b u t a n o l : a c e t i c acid:water = 4:1:5 -r e v e a l e d only one, f a s t - r u n n i n g p r i n c i p l e . 72 A p o s s i b l e reason f o r the discrepancy between the r e s u l t s of d i f f e r e n t authors may have been that at some times one p a r t i c u l a r p r i n c i p l e occurred alone, w h i l s t at other times v a r i o u s intermediate mixtures of the two were present - mixtures dependent on unknown f a c t o r s such as sex, season, or l o c a t i o n . Since i n a d d i t i o n to t h e i r chromato-graphic r e s o l u t i o n , E^ and E^ were a l s o d i s t i n g u i s h e d by t h e i r d i f f e r e n t degrees of magnesium p o t e n t i a t i o n , i t was p o s s i b l e that the presence- of two such p r i n c i p l e s i n v a r y i n g p r o p o r t i o n s might have been detected by a study of the v a r i a b i l i t y i n the magnesium p o t e n t i a t i o n s of crude neurointermediate lobe e x t r a c t s . An attempt was made to determine whether there was such a v a r i a b i l i t y i n the magnesium p o t e n t i a t i o n s of twelve e x t r a c t s of Raia r h i n a p i t u i t a r i e s , obtained i n d i f f e r e n t t r a w l s at v a r i o u s l o c a t i o n s . The neurointermediate lobes from samples 1-12 (Appendix I) were e x t r a c t e d and assayed f o r o x y t o c i c a c t i v i t y on the r a t uterus both i n the presence and absence of magnesium ions. T h e i r a c t i v i t i e s and the r e s u l t i n g magnesium p o t e n t i a t i o n s are l i s t e d i n Table IV. The p o t e n t i a t i o n s v a r i e d from 1.8 ± 0.4 to 17.5 ± 4.4, and could have supported the e x i s t e n c e of v a r i o u s mixtures of two p e p t i d e s . However the range was unexpectedly wide. The known p o t e n t i a t i o n s of E^ and E^ d i f f e r e d only from 2 to 6 '(.Table I I I , page 33), and the highest p o t e n t i a t i o n s which had been r e p o r t e d f o r neurophypophysial p r i n c i p l e s were Table IV 7 3 Oxytocic A c t i v i t i e s , with and without Magnesium, of Crude Neurointermediate Lobe E x t r a c t s of Raia r h i n a ; and t h e i r Magnesium P o t e n t i a t i o n s Sample Rat uterus a c t i v i t y Magnesium number 8 , (mU/mg d r i e d t i s s u e ) p o t e n t i a t i o n 0 without Mg + + with Mg + + 1 2 . 2 + 0.3 k.o + 0.7 1.8 + 0.4 2 1.2 + 0.05 3.2 + 0.2 2.7 + 0.2 3 1.7 + 0.1 5.7 + 1.0 3.5 + 0.6 k • 2.7 + 0.9 9.7 + 3.9 3.6 + 1.8 5 1.7 + 0.2 6.1 + 0.8 3.7 + 0.7 6 2.0 + O.k 10.9 + 1.7 5.6 + 1.2 7 0 . 9h + 0 .08 5.3 + 0.8 5.6 + 1.0 8 1.3 + 0.5 7.8 + 1.1+ 6.1 + 2.6 9 0 . 91 + 0 .11 7.0 + 0.7 7.6 + 1.1 10 l.k + 0.2 17-6 + 3.7 12. k + 2.8 11 2.2 + 0.4 37- 5 + 5.8 16.8 + 3.U 12 0.92 + 0.18 16.1 + 2.9 17.5 + k.k aAs i n Appendix I. Assays c a l c u l a t e d by the l+-point method of Holton ( 1 9 ^ 8 ) ; whenever three or more groups were obtained the f i d u c i a l l i m i t s are expressed, P = 0.05. Magnesium p o t e n t i a t i o n = ra t uterus a c t i v i t y , i n presence of 0.5 mM Mg + + r a t uterus a c t i v i t y , i n absence of Mg + + 74 R., =10 f o r g l u m i t o c i n (Acher et a l . , 1965) and R l f =10.8 f o r Mg — — Mg the s y n t h e t i c analogue, 3-norvalme o x y t o c i n ( K r e j c i and Polacek, 1968). Despite the s u r p r i s i n g range of the poten-t i a t i o n s obtained f o r the e x t r a c t s , the e x i s t e n c e of v a r i a b l e mixtures remained a p o s s i b i l i t y . T h i s could be checked by chromatographic s e p a r a t i o n of the p r i n c i p l e s . b. Paper chromatography of e x t r a c t s with d i f f e r -ent magnesium p o t e n t i a t i o n values An attempt was made to show by paper chromatography that the d i f f e r e n t p o t e n t i a t i o n s which had been found f o r the e x t r a c t s i n the previous experiment r e f l e c t e d d i f f e r i n g peptide mixtures, and that each extreme i n p o t e n t i a t i o n r e f l e c t e d a preponderance of e i t h e r the slow-running or the f a s t - r u n n i n g p e p t i d e . A c c o r d i n g l y , samples 1, 7, and 11 (R =1.8, 5-6, and 16.8, r e s p e c t i v e l y ) were subjected to Mg paper chromatography i n n - b u t a n o l : a c e t i c acid:water = 4:1:5-Although there had been marked d i f f e r e n c e s between the magnesium p o t e n t i a t i o n s of the three e x t r a c t s , they a l l behaved i n a s i m i l a r manner upon chromatography ( F i g . 3). In each case a s i n g l e slow-running peak of o x y t o c i c a c t i v i t y was found, and the f r o n t d i d not extend f u r t h e r forward than an Rrp of 0.4. It i s probable that t h i s unexpectedly slow m i g r a t i o n r e s u l t e d from the l a r g e amounts of i n e r t m a t e r i a l which were a p p l i e d to the o r i g i n ; from 0.22 to 0.53 ml of e x t r a c t , which represented from 8.8 to 21.2 mg of dry t i s s u e and contained approximately 1 to 3 mg of Lowry p e p t i d e , had 75 F i g u r e 3. Descending paper chromatograms of three crude neurointermediate lobe e x t r a c t s of Raia rh i n a , on Whatman 3MM paper, i n n - b u t a n o l : a c e t i c a c i d : water = 4 : 1 : 5 , at room temperature. White columns = s y n t h e t i c o x y t o c i n (Syntocinon, Sandoz); hatched columns = o x y t o c i c substances of Raia r h i n a . Numbers w i t h i n the columns i n d i c a t e the magnesium p o t e n t i a t i o n s of the e l u a t e s . Loads are s t a t e d as mU o x y t o c i c a c t i v i t y = mU r a t uterus a c t i v i t y i n the absence of Mg + +. T-6 Syntocinon LOAD RECOVERY Oxytocic Equivalent Magnesium activity extracted potentiation (mU) dry tissue (mg) Raia rhina sample ** 1 Syntocinon Raia rhina sample **7 Syntocinon Raia rhina sample *M1 4 0 2 0 4 0 2 0 •8 -9 10 9-2 1-8 4 0 — 9 . 0 1 6 8 43°/o 41°/o 5 7 " / . 2 0 21.3 5 . 6 170°/o 46° /o 106°/o R F 77 been a p p l i e d to each o r i g i n . The recovery of a c t i v i t y from two of the chromato-grams (samples 7 and l l ) was unexpectedly h i g h , 106% and 170% of the a c t i v i t y which had been a p p l i e d . A s i m i l a r phenomenon had p r e v i o u s l y been observed f o r chromatograms of two other elasmobranchs, Squalus acanthias and S c y l i o r h i n u s c a n i c u l u s ( P i c k e r i n g , p e r s o n a l communication to Perks, i 9 6 0 ) . The r e c o v e r y of the s y n t h e t i c o x y t o c i n standard (Syntocinon) from the Raia r h i n a chromatograms was h3% to 57%. Although each e x t r a c t ran as a s i n g l e o x y t o c i c f r a c t i o n , i t was p o s s i b l e that a peptide mixture might have been p a r t i a l l y r e s o l v e d w i t h i n the s u p e r f i c i a l l y continuous peak - e s p e c i a l l y as the proper development of the chromato-gram may have been i n h i b i t e d by the high l e v e l of i n e r t m a t e r i a l i n the e x t r a c t s . An attempt was made to show t h i s by a s s e s s i n g the magnesium p o t e n t i a t i o n of the o x y t o c i c agent i n each Rp u n i t . The r e s u l t s are i n d i c a t e d i n F i g . 3. It was found that the o x y t o c i c peaks were u n i f o r m l y p o t e n t i a t e d throughout t h e i r l e n g t h ; there was no i n d i c a t i o n of the separa-t i o n of a h i g h l y p o t e n t i a t e d p r i n c i p l e from a l e s s p o t e n t i a t e d p r i n c i p l e , even on the chromatogram of sample 7 S where the i n t e r m e d i a t e value of the magnesium p o t e n t i a t i o n might have suggested the presence of a peptide mixture. However, i t was s u r p r i s i n g to f i n d t h a t the p o t e n t i a t i o n s of the e l u a t e s were d i f f e r e n t from those of the o r i g i n a l crude e x t r a c t s . 78-This might have suggested the chromatographic removal of an i n h i b i t o r y or an a c t i v a t i n g substance from the e x t r a c t , but the changes i n p o t e n t i a t i o n of the e l u a t e s were not a l l i n a c o n s i s t e n t d i r e c t i o n . The R., f o r samples 7 and 11 had Mg decreased from 5.6 to 1.6 and from l6.8 to 3.7, r e s p e c t i v e l y , while sample 1 had i n c r e a s e d from 1.8 to 5.3. c. J o i n t assays of e x t r a c t s with d i f f e r e n t  magnesium p o t e n t i a t i o n values The v a r i a t i o n s which were observed i n the p o t e n t i a t -ions of the three e x t r a c t s before and a f t e r chromatography, and i n the p o t e n t i a t i o n s of the s e r i e s of twelve e x t r a c t s , may have been due to d i f f e r e n c e s between the u t e r i on which they were assayed. To i n v e s t i g a t e t h i s p o s s i b i l i t y , samples which had been found to have widely d i f f e r e n t p o t e n t i a t i o n s (Table I\V) were reassayed, t o g e t h e r , on the same u t e r i . Samples 1 and 11 were assayed " j o i n t l y " both i n the presence and absence of magnesium, as were samples 7 and 11 and samples 3 and 10. In a l l three cases the two " j o i n t " p o t e n t i a t i o n s were found to be s i m i l a r (Table 'V;). The p o t e n t i a t i o n s of samples 1 and 11 converged from the wide values of 1.8 ± 0.4 and l 6 . 8 ± 3.4, r e s p e c t i v e l y , to 4.8 and 4.1 ± 1.1. The p o t e n t i a t i o n s of samples 7 and 11, which were 5.6 ± 1.0 and 16.8 ± 3.4 on independent assays, became 2.3 ± 0.9 and 2.5 on j o i n t assay; and the values f o r samples 3 and 10, o r i g i n a l l y 3.5 ± 0.6 and 12.4 ± 2 . 8 , were found to be 6.7 ± 1 . 7 and 8.4. Table V i Magnesium P o t e n t i a t i o n s of P a i r s of Raia r h i n a Neurointermediate Lobe E x t r a c t s during Independent and J o i n t Rat Uterus Assays Experiment Sample number Independent d e t e r m i n a t i o n 3 . Act i v i t i e s Potent i a t i o n J o i n t d e termination A c t i v i t i e s P o t e n t i a t i o n As s ay mU/mg d r i e d RMg As s ay mU/ m£ d r i e d RMg t i s s u e t i s sue 1 1 RU C 2.2 + 0.. 3 RU 2.4 RU+Mg 4.0 + 0.7 1.8 ± 0 . 4 RU+Mg 11. 5 ± 2.5 4.8 11 RU 2.2 + 0.4 RU 3.1 i. 0.7 RU+Mg 37.5 + 5.8 16.8 + 3.4 RU+Mg 12.6 ± 2.8 4.1 ± l . 1 2 7 RU 0 .94 + 0.08 RU 1.8 ± 0.6 RU+Mg 5.3 + 0.8 5.6 ± 1.0 RU+Mg 4.1 ± 1.6 2.3 ± 0. 9 11 RU 2.2 + O.k RU 1.6 RU+Mg 37.5 + 5.8 16. 8 ± 3.4 RU+Mg 4.1 2.5 3 3 RU 1.7 + 0.1 RU 1.3 ± 0.2 RU+Mg 5-7 + 1.0 3.5 ± 0.6 RU+Mg 8.4 + 1.9 6.7 ± 1. 7 10 RU 1.4 + 0.2 RU 1.3 RU+Mg 17.6 + 3.7 12. k ± 2 . 8 RU+Mg 11. 2 + 4.9 8.4 -<1 Table V i continued aAs i n Table IV. ^Assays c a l c u l a t e d by the 4-point method of Holton ( 1 9 ^ 8 ) ; whenever three or more groups were obtained the f i d u c i a l l i m i t s are expressed, P = 0.05. c A b b r e v i a t i o n s used: RU = assay performed on r a t u t e r u s , i n absence of magnesium i o n . RU+Mg = assay performed on r a t u t e r u s , i n presence of 0.5 mM magnesium i o n . R M = magnesium p o t e n t i a t i o n , as i n Table IV. oo o These changes i n p o t e n t i a t i o n r e f l e c t e d changes i n the potency of the e x t r a c t s on both types of assay (see Table V)'; they could not be a s c r i b e d s o l e l y to i n c o n s i s t e n c i e s i n the assay values produced i n the presence of Mg + +, nor i n i t s absence. d. The i n f l u e n c e of the estrous c y c l e on  magnesium p o t e n t i a t i o n values The responses of d i f f e r e n t u t e r i to the same p r i n c i p l e might be i n f l u e n c e d by t h e i r stage i n the estrous c y c l e . The u t e r i used i n these experiments had a l l been taken from r a t s i n the estrous s t a t e , as determined by v a g i n a l smear, but no attempt had been made to d i s t i n g u i s h between e a r l y and l a t e e s t r o u s . Therefore sample 6 was reassayed, both with and without magnesium, on the two horns of a uterus taken from a r a t i n e a r l y e s t r o u s , and then again on the horns of a uterus i n l a t e e s t r o u s . While the potency of the e x t r a c t d i f f e r e d s i g n i f i c a n t l y from e a r l y estrous to l a t e estrous when the assays were performed i n the presence of magnesium i o n s , the two r e s u l t i n g p o t e n t i a t i o n s were not s i g n i f i c a n t l y d i f f e r e n t from each o t h e r , and both were w e l l w i t h i n the range of values observed e a r l i e r (1.8 ± 0.4 to IT.5 ± 4.4). The pot e n c i e s found on the e a r l y estrous p r e p a r a t i o n were 3.2 ± 0.8 mU/mg d r i e d t i s s u e without magnes-ium and 25.9 ± 6.6 mU/mg when magnesium was pre s e n t . On the l a t e estrous p r e p a r a t i o n the p o t e n c i e s were 2.6 ± 0.4 mU/mg without magnesium and l4.2 ± 2.3 mU/mg with magnesium. The r e s u l t i n g p o t e n t i a t i o n s were 8.2 ± 3.0 and 5.5 ± 1.3 f o r e a r l y and l a t e e s t r o u s , r e s p e c t i v e l y . Therefore these b r i e f 82 experiments suggest that the d i f f e r e n c e between u t e r i i n e a r l y and i n l a t e estrous cannot account f o r the f u l l range of p o t e n t i a t i o n s which were observed. Only f u r t h e r exten-s i v e work could e s t a b l i s h any more s u b t l e r e l a t i o n s h i p s which might e x i s t , and i t was considered more important to make more d i r e c t a t t a c k s on the chemistry and pharmacology of the skate p r i n c i p l e s at t h i s stage. 3. Comparison of Crude E x t r a c t with S y n t h e t i c  Glumitocin G l u m i t o c i n , the p r i n c i p l e o c c u r r i n g i n s e v e r a l species of skate (Acher e_t a l . , 1965 ; 1967 ; Chauvet et_ a l . , 1965), was s y n t h e s i z e d during the course of t h i s study (Manning e_t a l . , 1968) . This made i t p o s s i b l e to compare the Raia r h i n a p r i n c i p l e to a s y n t h e t i c peptide with which i t might w e l l be i d e n t i c a l . a. Assays of crude e x t r a c t against g l u m i t o c i n Thirty-two neurointermediate lobes from sample 22 (Appendix I) were e x t r a c t e d and assayed against s y n t h e t i c g l u m i t o c i n f o r r a t uterus a c t i v i t y both with and without magnesium, f o r m i l k - e j e c t i o n a c t i v i t y , and f o r a n t i d i u r e t i c a c t i v i t y . The r e s u l t s are shown i n Table VI, and the r a t i o s of the a c t i v i t i e s are expressed as D„, , D,,^ , and D. _ T T to * Mg' ME' ADH i n d i c a t e t h a t the assays were performed against a standard ( i n t h i s case, s y n t h e t i c g l u m i t o c i n ) other than o x y t o c i n (Syntocinon) or v a s o p r e s s i n ( P i t r e s s i n ) . Table VI A c t i v i t i e s and A c t i v i t y Ratios of Crude Raia r h i n a Neurointermediate Lobe E x t r a c t , Assayed Against S y n t h e t i c G l u m i t o c i n Sample A c t i v i t i e s number 3 - (ng glumitocin/mg d r i e d t i s s u e ) A c t i v i t y r a t i o s RU C RU+Mg ME ADH D Ms 'ME D ADH 22 294 ± 39 233 ± 25 l ) d 3 6 2 (32 NIL) 2) Ul3 ± 107 16U0 0.79 ± O.lU 1 ) 1 . 2 5.6 2) 1.1+ ± 0.1+ aAs i n Appendix I. ^Assays c a l c u l a t e d by the U-point method of Holton ( 1 9 ^ 8 ) ; whenever three or more groups were obtained the f i d u c i a l l i m i t s are expressed, P = 0.05. A b b r e v i a t i o n s used: RU = a c t i v i t y on r a t u t e r u s , i n absence of magnesium i o n s . RU+Mg = a c t i v i t y on r a t u t e r u s , i n presence of 0.5 mM Mg + +. ME = a c t i v i t y on rab-b i t m i l k - e j e c t i o n assay. ADH = a c t i v i t y on r a t a n t i d i u r e t i c assay. A c t i v i t y r a t i o s , D^g, D^g , and D/^TJH > a r e "the r a t i o s of the a c t i v i t y found on each assay (RU+Mg, ME, and ADH) to the a c t i v i t y found on the r a t uterus assay (RU); where D i n d i c a t e s that the assays were performed against a peptide other than the normal standards. ^Two separate determinations were made f o r m i l k - e j e c t i o n a c t i v i t y . 8 4 . The p o t e n c i e s of the e x t r a c t on the three assays used p r i m a r i l y f o r n e u t r a l p r i n c i p l e s ( r a t uterus with magnesium, r a t uterus without magnesium, and m i l k - e j e c t i o n ) were s i m i l a r ; the magnesium p o t e n t i a t i o n was e l i m i n a t e d when g l u m i t o c i n r e p l a c e d o x y t o c i n as a standard, and the two a c t i v i t y r a t i o s , D„ and D.,„, d i d not d i f f e r s i g n i f i c a n t l y from one. This Mg ME' B J was c o n s i s t e n t with the i d e n t i t y of the Raia r h i n a p r i n c i p l e with g l u m i t o c i n . However the e x t r a c t was much more a c t i v e than g l u m i t o c i n on the a n t i d i u r e t i c assay. This could be ex p l a i n e d by the presence i n the crude e x t r a c t of a second p e p t i d e , which had a much gr e a t e r a n t i d i u r e t i c a c t i v i t y than that of g l u m i t o c i n . During subsequent p u r i f i c a t i o n of the a c t i v e p r i n c i p l e s a b a s i c f r a c t i o n having 209 mU of a n t i -d i u r e t i c a c t i v i t y was separated away from the n e u t r a l f r a c t i o n of 1008 mU r a t uterus a c t i v i t y . The b a s i c f r a c t i o n was not c h a r a c t e r i z e d f u r t h e r , but was t e n t a t i v e l y regarded as a r g i n -ine v a s o t o c i n ( S e c t i o n C3b, page 1954) • Table VI!"; shows the a c t i v i t y r a t i o s t h at would be expected from a mixture of g l u m i t o c i n (1008 mU r a t uterus a c t i v i t y , no Mg + + present) with a r g i n i n e v a s o t o c i n (209 mU a n t i d i u r e t i c a c t i v i t y ) . The experimental value of D A D H = 5.6 agreed w e l l with the t h e o r e t i c a l value of D ^ D J J = 5.0, w h i l s t the observed values f o r and D^ -g remained c o n s i s t e n t with the expected v a l u e s . C a l c u l a t i o n shows t h a t the data could a l s o apply to the presence of g l u m i t o c i n with e i t h e r of the two other n a t u r a l l y o c c u r r i n g b a s i c p e p t i d e s , a r g i n i n e v a s o p r e s s i n or l y s i n e 8 5 . Table VII A Comparison of the A c t i v i t y R a t i o s found f o r Raia r h i n a Neurointermediate Lobe E x t r a c t when Assayed against Syn-t h e t i c G l u m i t o c i n , to the T h e o r e t i c a l A c t i v i t y R a t i o s Expected from Mixtures of Gl u m i t o c i n and Small Amounts of Ba s i c Peptide Type of data Peptide•mixture A c t i v i t y r a t i o s Mg ME ADH Experimental Raia r h i n a , 0.7.9 1 ) 1 . 2 crude e x t r a c t ± 0 . 1 4 2 ) 1 . h±o. k 5 . 6 T h e o r e t i c a l c G l u m i t o c i n with 0 . 9 2 d 1 ) e o . 9 3 5 . 0 a r g i n i n e v a s o t o c i n 2 ) 0 . 9 2 Glumitocin with T. 0 0 1 ) 1 . 0 0 5 . 5 a r g i n i n e v a s o p r e s s i n 2) 1 . 0 0 G l u m i t o c i n with 1 . 0 0 1} 1 . 0 0 5 . h l y s i n e v a s o p r e s s i n 2} 1 . 0 0 G l u m i t o c i n with 0 . 6 8 1] 0 . 7 1 3 . 3 8 - l y s i n e o x y t o c i n 2) 0 . 6 6 A b b r e v i a t i o n s as i n Table VI. A c t i v i t y r a t i o s f o r crude e x t r a c t are a l s o given i n Table • T h e o r e t i c a l peptide mixtures c o n s i s t of g l u m i t o c i n ( 1 0 0 8 mU r a t uterus a c t i v i t y without Mg + + present) and b a s i c pep-t i d e ( 2 0 9 mU r a t a n t i d i u r e t i c a c t i v i t y ) , where the b a s i c peptide i s one of a r g i n i n e v a s o t o c i n ( 8 - a r g i n i n e o x y t o c i n ) , a r g i n i n e v a s o p r e s s i n (3-phe, 8 - a r g o x y t o c i n ) , l y s i n e vasopre s i n (3-phe, 8 - l y s o x y t o c i n ) , or 8 - l y s i n e o x y t o c i n . T h e o r e t i c a l a c t i v i t y r a t i o s were c a l c u l a t e d from the f o l -lowing data. References are i n d i c a t e d i n brackets and l i s t e d below. A r g i n i n e v a s o t o c i n : R M g = 1 . 9 ( a ) , R M E = 2 . 2 ( a ) , R A D H = 2 . 0 ( b ) . A r g i n i n e v a s o p r e s s i n : R M g = 2 . 3 ( a ) , R M E = 5 . 7 ( a ) , R A r ) H=4 U ( a.) . Lysine v a s o p r e s s i n : R M ^ = 2 . 5 ( a ) , R^E = 1 0 . 2 ( a) , R D^g=.3.3 (-a.) . 8 - l y s i n e o x y t o c i n : R„ = 1 . 9 ( a ) , R M F = 2 . 3 ( a) , R A T m = 0 ...31( a) . 86 Table V I I c o n t i n u e d G l u m i t o c i n : R M g = 9 - 5 ( c ) , R M E = ( l ) 8 . 2 ( c ) , and (2) 1 5 . 6 ( c ) , E A B E = 0 . 0 k 6 ( d ) . R e f e r e n c e s : (a) Sawyer (1965), (b) Sawyer et a l . (1961), (c) t h i s t h e s i s , T able V I I I , (dT Sawyer e_t a l . (1969). eThe two t h e o r e t i c a l v a l u e s of D ^ E l i s t e d f o r each pep-t i d e m i x t u r e c o r r e s p o n d t o the two v a l u e s found f o r the Rj£g of s y n t h e t i c g l u m i t o c i n on two s e p a r a t e assays (see Table V I I I ) , and a l so t o the two v a l u e s of Djyj-g found f o r the crude e x t r a c t on t h e same two assays (Table V I ) . 87 v a s o p r e s s i n , or to a mixture of g l u m i t o c i n w i th the p u r e l y s y n t h e t i c b a s i c p e p t i d e , 8 - l y s i n e o x y t o c i n (Tab le v l l ) . However the p resence of these b a s i c p r i n c i p l e s i s u n l i k e l y . The v a s o p r e s s i n s have been found to be c o n f i n e d to the mammals, and perhaps the b i r d s ; 8 - l y s i n e o x y t o c i n has low a n t i d i u r e t i c a c t i v i t y , and has not been found i n n a t u r e . b. Assays of crude e x t r a c t s and g l u m i t o c i n a g a i n s t  o x y t o c i n Dur ing each of the p r e c e e d i n g ra t u t e r u s and m i l k -e j e c t i o n a s s a y s , one group of responses was o b t a i n e d which i n c l u d e d the responses to a h i g h and a low dose o f s y n t h e t i c o x y t o c i n ( S y n t o c i n o n ) . The a c t i v i t i e s of the e x t r a c t and of g l u m i t o c i n were e s t i m a t e d r e l a t i v e to t h i s more u s u a l s t a n d a r d , but the assays were not s u f f i c i e n t l y extended to g ive v a l u e s f o r the e r r o r s of these e s t i m a t e s . The a c t i v i t i e s and t h e i r r a t i o s are l i s t e d i n Tab le V I I I . It can be seen tha t two d i f f e r e n t m i l k - e j e c t i o n p o t e n c i e s were found f o r both the crude e x t r a c t and g l u m i t o c i n , even though t h e i r a c t i v i t i e s r e l a t i v e to each o ther were s i m i l a r on the two assays (Tab le VI)-. T h i s i s p a r t i c u l a r l y n o t a b l e , s i n c e both assays were per formed on the same r a b b i t at a twelve day i n t e r v a l ; d e s p i t e t h i s the potency found f o r both unknowns was 100% g r e a t e r on the second a s s a y . S ince the second assay suggested an i n c r e a s e i n p o t e n c y , the r e s u l t s cannot be e x p l a i n e d by d e t e r i o r a t i o n of the unknowns. The s tandards were p r e p a r e d f r e s h l y . 88; Table VIII A c t i v i t i e s and A c t i v i t y R a t i o s of Raia r h i n a Neuro-int e r m e d i a t e Lobe E x t r a c t and of S y n t h e t i c G l u m i t o c i n , Assayed against S y n t h e t i c Oxytocin (Syntocinon) P r i n c i p l e A c t i v i t i e s 8 , Act i v i t y (mU/mg d r i e d t i s s u e ) r at i o s or (mU/mg gl u m i t o c i n ) RU b RU+Mg ME R R Mg ME. E x t r a c t of Raia r h i n a 2.7 16.3 l ) C 22.5 6.0 l ) 8.3 2) U7.3 2) 17.5 Glum i t o c i n 7602 72 ,1+1+6 l ) 62 ,196 9-5 l ) 8.2 2) 118,860 2) 15.6 a A l l values were obtained by the l+-point method of Holton (191+8); only s i n g l e groups were obtained d u r i n g these experiments which were designed to compare the e x t r a c t with g l u m i t o c i n , and t h e r e f o r e f i d u c i a l l i m i t s c o uld not be c a l c u l a t e d . A b b r e v i a t i o n s as i n Table V l ; . a except the a c t i v i t y -r a t i o s , R Mg and Rj^g; where R i n d i c a t e s that the assays were performed against a normal standard ( s y n t h e t i c o x y t o c i n ) . cTwo m i l k - e j e c t i o n assays were performed (see Table VII, footnote e ) . 89 The corresponding r a t i o s of m i l k - e j e c t i o n to r a t uterus a c t i v i t y ( R M E ^ r e f l e c t e d these d i f f e r e n c e s i n m i l k -e j e c t i o n potency with values of 8 . 2 and 15.6 f o r g l u m i t o c i n , and 8 . 3 and 17.5 f o r the e x t r a c t . E a r l i e r i n t h i s i n v e s t i -g a t i o n a t h i r d value of R,.,-, = h.k had a l s o been found f o r an ME e x t r a c t from t h i s species ( S e c t i o n B l , page 7 0 ) . V a r i a t i o n i n the r a t i o of m i l k - e j e c t i o n a c t i v i t y to r a t uterus a c t i v i t y had not p r e v i o u s l y been r e p o r t e d f o r any neurohypophysial p r i n c i p l e , but the values found here, which ranged from h.k to 17-5 f o r crude e x t r a c t s of R_. r h i n a , and from 8 . 2 to 15.6 f o r g l u m i t o c i n , demonstrate again the danger inherent i n comparing assay values obtained on d i f f e r e n t p r e p a r a t i o n s , when the unknown and standard are of d i f f e r e n t molecular s t r u c t u r e . C. The P u r i f i c a t i o n of the E x t r a c t Pharmacological s t u d i e s of the e x t r a c t of Raia r h i n a ( S e c t i o n B 3, page 82) i n d i c a t e d t h a t i t contained g l u m i t o c i n . An attempt was made to p u r i f y and analyze the p r i n c i p l e so that i t s amino a c i d composition could be compared to the known composition of g l u m i t o c i n . 1. E x t r a c t i o n of the Glands, and P u r i f i c a t i o n of  the E x t r a c t by Gel F i l t r a t i o n For p u r i f i c a t i o n procedures, the glands from samples 13 to 2h (Appendix I) were pooled, with the exception of the 32 glands which were e x t r a c t e d f o r comparison with 90 g l u m i t o c i n ( S e c t i o n B3, page 82). The 220 d r i e d n e u r o i n t e r -m e d i a t e l o b e s , w e i g h i n g 1917 mg, were e x t r a c t e d i n 48 ml o f 0.25% a c e t i c a c i d and f i l t e r e d on a B u c h n e r f u n n e l . The r e s i d u e w h i c h r e m a i n e d a f t e r f i l t r a t i o n was r e - e x t r a c t e d i n 15 ml o f 0.25% a c e t i c a c i d and r e f i l t e r e d . A t o t a l o f 57 ml e x t r a c t was r e c o v e r e d . The o x y t o c i c a c t i v i t y ( r a t u t e r u s , w i t h o u t magnesium) o f t h e e x t r a c t was low, 36.8 ± 6.9 mU/ml, or 1.1 mU/mg d r i e d t i s s u e . I t s Lowry p e p t i d e c o n t e n t was 5,100'ug/ml and t h e r a t i o o f o x y t o c i c a c t i v i t y t o Lowry p e p t i d e was J. 2 mU/mg. The t o t a l a c t i v i t y i n t h e e x t r a c t was 2098 mU. To t h i s was added 8 ml o f e x t r a c t r e m a i n i n g f r o m p r e v i o u s e x p e r i m e n t s and c o n t a i n i n g 392 mU o x y t o c i c a c t i v i t y , t o g i v e a t o t a l o f 2490 mU o f o x y t o c i c a c t i v i t y . The e x t r a c t was p a s s e d t h r o u g h a 90 cm x 2.4 cm column o f Sephadex G-15 g e l ( i n 0.2 M a c e t i c a c i d ) i n two l o t s o f 30 and 35 ml e a c h . The l a r g e sample s i z e was a d o p t e d i n o r d e r t o m i n i m i z e t h e d i l u t i o n o f t h e e x t r a c t d u r i n g i t s p a s s a g e t h r o u g h t h e column. The r e s u l t s o f t h e two run s a r e summarized i n T a b l e XII,I,page 124). The a c t i v e e l u a t e s f r o m t h e two r u n s were combined i n a volume o f 284 ml. T h i s had an o x y t o c i c a c t i v i t y o f 7.2 mU/ml and a Lowry p e p t i d e c o n t e n t o f 266 pg/ml. The t o t a l a c t i v i t y r e c o v e r e d was 2,045 mU or 82% o f t h a t a p p l i e d t o t h e column. A p p r o x i m a t e l y 77% o f t h e o r i g i n a l Lowry p e p t i d e had been s e p a r a t e d away f r o m t h e n e u r o h y p o p h y s i a l p r i n c i p l e s , and t h e r a t i o o f o x y t o c i c 91 a c t i v i t y to Lowry peptide i n the elua t e was 27 mU/mg, a 3-8 f o l d p u r i f i c a t i o n . The sample was d i l u t e d to k.k times i t s o r i g i n a l volume. 2. Passage of the E x t r a c t through Ion R e t a r d a t i o n  Resin and DEAE Sephadex The a c t i v e eluate from the Sephadex G-15 was passed through a ^0 cm x 0.9 cm column of Biorad Ion R e t a r d a t i o n Resin A G 1 1 A 8 , which removed some of the s a l t content from the e l u a t e and so f a c i l i t a t e d the a d s o r p t i o n of the a c t i v e p r i n c i p l e s onto a subsequent c a t i o n exchanger. The a c e t i c a c i d ( 0 . 2 M, s p e c i f i c c o n d u c t i v i t y 0.71 mmho/cm), by which the peptides had been e l u t e d from the Sephadex G-15, was not removed by t h i s procedure. Nevertheless the s p e c i f i c c o n d u c t i v i t y of the peptide s o l u t i o n was reduced to 0.97 mmho/cm from 1 . 5 0 mmho/cm. There was no l o s s of o x y t o c i c act i v i t y . The e l u a t e from the ion r e t a r d a t i o n r e s i n was adjusted to pH 7-0 and s p e c i f i c c o n d u c t i v i t y 0 . 2 1 mmho/cm by dropwise a d d i t i o n of NH^OH and d i l u t i o n with water to a f i n a l volume fo ^ 5 0 0 ml. It was passed through a 35 cm x 0.9 cm column of DEAE Sephadex, an anion exchanger, which had been e q u i l i b r a t e d with 0 . 0 0 2 M NH^acetate (pH 7 - 0 ) . The a c t i v e p r i n c i p l e s were not adsorbed onto the g e l . How-ever t h i s step had been found necessary f o r the subsequent a d s o r p t i o n of the ox y t o c i c p r i n c i p l e of the skate Raia 92 oc e l l a t a onto CM Sephadex (Perks and Sawyer, 1 9 6 5 ) . No l o s s of o x y t o c i c a c t i v i t y was expected, but the l e v e l of a c t i v i t y i n the e l u a t e was too low to be assessed. 3. Chromatography of the P r i n c i p l e s on CM Sephadex a. The passage through the column The e l u a t e from the DEAE Sephadex was adjusted to pH 5 . 0 and s p e c i f i c c o n d u c t i v i t y Q . l 6 mmho/cm by dropwise a d d i t i o n of a c e t i c a c i d and d i l u t i o n with water to a f i n a l volume of 7 3 0 0 ml. It was a p p l i e d to a 35 cm x 0 . 9 cm column of CM Sephadex, Lot 8 8 5 2 , which had been e q u i l i b r a t e d with 0 . 0 0 2 M JHj^acetate, pH 5 . 0 . The neurohypophysial p r i n c i p l e s were adsorbed onto the exchanger. They were e l u t e d with HH^acetate b u f f e r which i n c r e a s e d stepwise i n c o n c e n t r a t i o n from 0 . 0 0 2 M (pH 5 . 0 ) to 0 . 1 5 M (pH 5 . 0 ) and then to 0 . 2 0 M (pH 7 . 0 ) . Three f r a c t i o n s of b i o l o g i c a l a c t i v i t y were e l u t e d from the column. The peaks of a c t i v i t y were not assayed throughout t h e i r l e n g t h . This was i n order to conserve the small amount of neurohypophysial p r i n c i p l e i n the e l u a t e s , and to avoid i n t r o d u c i n g contamination which might have been s i g n i f i c a n t i n r e l a t i o n to the amount of a c t i v e p e p t i d e . The beginning and- end of each f r a c t i o n was l o c a t e d , and i t s t o t a l a c t i v i t y and p o s i t i o n i n the e l u a t e i s i n d i c a -t e d i n Figure ha. 93 F i g u r e ha.. Chromatogram of the p a r t i a l l y p u r i f i e d n e u r o i n t e r m e d i a t e l o b e e x t r a c t of R a i a r h i n a on a 35 cm x 0.9 cm column of. CM Sephadex. The a c t i v i t y . (1,827 mU r a t . u t e r u s a c t i v i t y , no M g + + p r e s e n t ) was a p p l i e d i n 7 » 3 0 0 ml of s o l u t i o n at pH 5 .0 and. s p e c i f i c c o n d u c t i v i t y 0 . 1 6 mmho/cm, and was e l u t e d wi th NH^acetate b u f f e r of i n c r e a s i n g c o n c e n t r a t i o n , , from 0.002 M (pH 5.0):.' 0.15 M NH^acetate (pH 5.0) was i n t r o d u c e d s tepwise at tube 5, and 0.20 M NH^acetate (pH 7.0) was i n t r o d u c e d s tepwise at tube 1+7. Empty c i r c l e s =. s p e c i f i c conduc -t i v i t y of the e l u a t e . Hatched columns = r a t u t e r u s a c t i v i t y , wi thout M g + + : . f r a c t i o n 1 = 356 mU; f r a c t i o n 2 = 652 mU. C r o s s -hatched column = a n t i d i u r e t i c a c t i v i t y : f r a c t i o n 3 = 209 mU. Volume of tubes 1-1+7 = 1+.6 m l , of tubes 1+8-90 = 3.5 m l . F i g u r e 1+b. Chromatogram of combined f r a c t i o n 1 . and f r a c -t i o n 2 of R a i a r h i n a ( p u r i f i e d on Sephadex G-15 and on CM Sephadex - see F i g . 1+a, above) on a second 35 cm x 0.9 cm column of CM Sephadex. The a c t i v i t y was a p p l i e d i n 150 ml of s o l u t i o n at pH 5.0. and. s p e c i f i c c o n d u c t i v i t y 1.6 mmho/cm, and was e l u t e d wi th NH^acetate b u f f e r of i n c r e a s i n g c o n c e n t r a t i o n , from 0.022 M (pH 5 - 0 ) : 0.20 M NH^acetate (pH 5.0) was i n t r o d u c e d i n t o the b u f f e r r e s e r v o i r at tube 3. Empty c i r c l e s = s p e c i f i c c o n d u c t i v i t y of the e l u a t e . Hatched column = o x y t o c i c a c t i v i t y ( r a t u t e r u s a c t i v i t y , w i thout M g + + ) ; 1+39 mU. Tube volumes = 1+. 6 m l . 94 95 Two of the f r a c t i o n s were e l u t e d s e p a r a t e l y during the course of the wash with 0.15 M b u f f e r (pH 5.0); t h i s f o l l o w e d the behavior which had been found f o r the n e u t r a l p e p t i d e , o x y t o c i n (Syntocinon), during p r e l i m i n a r y e x p e r i -ments. The t h i r d f r a c t i o n was e l u t e d at pH 7.0 by 0.20 M NH^acetate. F r a c t i o n 1, tubes 9-17 i n c l u s i v e , contained 356 mU of r a t uterus a c t i v i t y i n a combined volume of 4l.4 ml. F r a c t i o n 2, tubes 24-35 i n c l u s i v e , contained 652 mU r a t uterus a c t i v i t y i n 55.2 ml. The t h i r d f r a c t i o n , tubes 73 - 8 4 , had an a n t i d i u r e t i c a c t i v i t y of 209 mU i n 38 ml (Table XIV, page 1 3 4 ) . b. The b a s i c f r a c t i o n The t h i r d f r a c t i o n of a c t i v i t y was detected by i t s a n t i d i u r e t i c a c t i v i t y . I t was e l u t e d from the g e l i n the same p o s i t i o n as the b a s i c peptide from Squalus acanthias ( F i g . 9, page 133; a l s o see Sawyer, 1967). The Squalus p r i n c i p l e was i d e n t i f i e d as a r g i n i n e v a s o t o c i n . A r g i n i n e v a s o t o c i n occurs i n other elasmobranch s p e c i e s , i n c l u d i n g skate (Acher et a l . , 1965 ; 1967; Chauvet et_ aJL . , 1965 ; Sawyer, 1 9 6 7 ) 9 and has been found to be the b a s i c peptide i n many non-mammalian v e r t e b r a t e s (Table I I , page 1 3 ) . On t h i s p r e l i m i n a r y b a s i s the t h i r d f r a c t i o n from Raia r h i n a was t e n t a t i v e l y regarded as a r g i n i n e v a s o t o c i n . 9 6 c. Rechromatography of the combined n e u t r a l  f r a c t i o n on CM Sephadex During e a r l i e r p u r i f i c a t i o n s of the neurohypophysial p r i n c i p l e s produced by the d o g f i s h Squalus a c a n t h i a s , i t had been found that two n e u t r a l f r a c t i o n s were e l u t e d from CM Sephadex, i n the same p o s i t i o n s i n the e l u a t e as f r a c t i o n s 1 and 2 found here f o r Raia r h i n a ( F i g . 9 s page 133). Care-f u l comparison of the two Squalus f r a c t i o n s had i n d i c a t e d that t h e i r neurohypophysial peptide contents were i d e n t i c a l , and that i t was probable that the s p l i t t i n g of the n e u t r a l p r i n c i p l e on the r e s i n had r e s u l t e d from the high volumes i n which the samples had been a p p l i e d to the columns ( S e c t i o n I I C 7 5 page i h l ) . The Raia r h i n a p r i n c i p l e s had a l s o been loaded onto the r e s i n i n a high volume (7300 ml). Since p h armacological s t u d i e s of the Raia e x t r a c t had p o i n t e d to a s i n g l e n e u t r a l p e p t i d e , g l u m i t o c i n ( S e c t i o n B3, page 8 2 ) , i t seemed reasonable to suppose that f r a c t i o n 1 and f r a c t i o n 2 of t h i s s p e c i e s a l s o contained i d e n t i c a l p r i n c i p l e s . T h e r e f o r e , i n order to conserve m a t e r i a l f o r l a t e r amino a c i d a n a l y s e s , they were combined. F u r t h e r , i t was a n t i c i p a t e d , on the b a s i s of e a r l i e r s i m i l a r experiments with elasmobranch m a t e r i a l ( S e c t i o n IIC7g» page 15l), that i f the combined Raia r h i n a a c t i v i t y were a p p l i e d to the CM Sephadex column i n a reduced volume, then the " f r a c t i o n 1" p o r t i o n would be e l u t e d i n the p o s i t i o n of " f r a c t i o n 2 " , and contaminants which might be present i n 97 " f r a c t i o n l " would be separated away. In order to o b t a i n t h i s reduced volume, and because a l l the a c t i v i t y was ex-pected to be e l u t e d at the higher c o n d u c t i v i t y of f r a c t i o n 2, the s o l u t i o n to be a p p l i e d to the column was not d i l u t e d back to a s p e c i f i c c o n d u c t i v i t y of 0.16 mmho/cm, as i n the i n i t i a l CM Sephadex procedure, but only to the s p e c i f i c c o n d u c t i v i t y of 1.6 mmho/cm (pH 5-0). Because of these c o n s i d e r a t i o n s , the n e u t r a l f r a c t i o n s were t r e a t e d as f o l l o w s : The two n e u t r a l f r a c t i o n s from the i n i t i a l CM Sephadex p u r i f i c a t i o n were combined -into a t o t a l volume of 96.6 ml, which contained a t o t a l of 1008 mU ox y t o c i c a c t i v i t y ( r a t u t e r u s , without magnesium). This s o l u t i o n was l y o p h i l i z e d f o r the removal of ammonium b u f f e r s a l t s , and the powder was taken up i n 30 ml of weak b u f f e r s o l u t i o n (0.002 M NH^acetate, pH 5.0). This was then d i l u t e d to a s p e c i f i c c o n d u c t i v i t y of 1.6 mmho/cm, at pH 5.0, i n a f i n a l volume of 150 ml, which represented only 2% of the volume a p p l i e d i n the e a r l i e r CM Sephadex run. The s o l u t i o n was a p p l i e d to a 35 cm x 0.9 cm column of CM Sephadex, Lot 8852, which had been e q u i l i b r a t e d with 0.022 M NH^acetate (pH 5.0). The a c t i v i t y was adsorbed onto the g e l , and was e l u t e d on an NH^acetate gra d i e n t from 0.022 M (pH 5-0) to 0.20 M (pH 5.0). The o x y t o c i c a c t i v i t y appeared i n a s i n g l e f r a c t i o n between s p e c i f i c c o n d u c t i v i t i e s of 6.8 mmho/cm and 7.^ + mmho/cm (tubes 22-30 i n c l u s i v e , F i g . k\>) ; t h i s j u s t i f i e d the d e c i s i o n 98 to combine the two o x y t o c i c f r a c t i o n s , and again suggested that they contained the same p r i n c i p l e . A l l the tubes which contained a c t i v i t y were combined. The t o t a l a c t i v i t y was 439 mU, i n a volume of 33.5 ml. 4. Amino A c i d A n a l y s i s of the Oxytocic P r i n c i p l e The o x y t o c i c p r i n c i p l e of another skate, Raia  oc e l l a t a , had been p u r i f i e d by a s i n g l e e l u t i o n from CM Sephadex (Perks and Sawyer, 1965)» and i t was a n t i c i p a t e d that the n e u t r a l p r i n c i p l e from R_. r h i n a , which had been submitted twice to chromatography on t h i s same exchanger:, would a l s o be pure at t h i s stage. Therefore the s o l u t i o n c o n t a i n -ing the o x y t o c i c p r i n c i p l e was l y o p h i l i z e d f o r removal of b u f f e r s a l t s , and the p u r i f i e d peptide was hydrolyzed i n 6 N HC1 at 107°C f o r 18 hours. The amino a c i d content of the h y d r o l y s a t e was determined on a B i o c a l BC-200 Amino A c i d A n a l y z e r . Three analyses were made. The r e s u l t s are given i n Table IX. It can be seen that the p u r i f i c a t i o n of the n e u t r a l p r i n c i p l e was not complete. N e v e r t h e l e s s , a cautious i n t e r -p r e t a t i o n of the data was p o s s i b l e . The b a s i c amino acids -histamine, l y s i n e , and a r g i n i n e - were present i n the h y d r o l y -sate i n c o n s i d e r a b l e p r o p o r t i o n . However, the behavior of the peptide on CM Sephadex ( F i g . 4, a and b ) , and the low r a t i o of a n t i d i u r e t i c to o x y t o c i c a c t i v i t y of the crude e x t r a c t (R A T- U = 0.02; S e c t i o n B l , page 70) had i n d i c a t e d T A B L E IX Amino Acid Analyses of the Purified Neutral Principle from Raia rhina Amino Acid Molar ratio to glutamic acid Sample l a Raia rhina principle Sample 2 Sample 3 Average values Glumitocin Neutral and acidic amino acids Cysteic acid 0.47 0.43 0.67 0.52 2 Aspartic acid 1.23 1.19 1.09 1.17 1 Serine 0.76 0.62 1.76 0.71 1 Glutamic acid 1.00 1.00 1.00 1.00 1 Proline 1.03 1.02 1.16 1.07 1 Glycine 1.84 1.76 1.90 1.83 1 Isoleucine 0.58 0.54 0.62 0.58 1 Tyrosine 0.76 0.85 1.38 1.00 1 Threonine 0.32 0.24 0.32 0.29 0 Alanine 0.25 0.35 0.33 0.31 0 Valine 0.39 0.38 0.41 0.39 0 Methionine 0.88 0.85 0.95 0.89 0 Leucine 0.34 0.29 0.38 0.34 0 Phenylalanine 0.13 0.34 0.53 0.33 0 Basic amino acids Histamine 1.65 0.85 2.19 1.56 0 Lysine +veb +ve +ve +ve 0 Arginine 0.43 0.39 4.17 1.66 0 Ammonia +ve +ve +ve +ve 4 Total amino acids Micromoles per unit oxytocic activity Neutral and acidic amino acids 2.356 2.566 2.260 2.394 -a A l l samples were from one hydrolysate of CM Sephadex-purified material. Basic, neutral and acidic amino acids were determined from each sample. Sample 1=88 mU oxytocic activity (no Mg"1-1" present). Sample 2=88 mU. Sample 3=264 mU. +ve: present in hydrolysate; molar ratio not calculated. 100 that b a s i c r e s i d u e s d i d not form part of i t s molecule; a s i m i l a r s i t u a t i o n has been r e p o r t e d from the c l o s e l y p a r a l l e l study of the n e u t r a l p r i n c i p l e of the skate, Raia o c e l l a t a, and the b a s i c r e s i d u e s were shown to be e x t r i n s i c to the a c t i v e molecule (Perks and Sawyer, 1965). It had been found duri n g previous p u r i f i c a t i o n s of the Squalus n e u t r a l f r a c t i o n t h a t some b a s i c amino acids were s t i l l present i n the pre-p a r a t i o n a f t e r i t s e l u t i o n from CM Sephadex, but these could be s u c c e s s f u l l y removed by chromatography on SE Sephadex ( S e c t i o n IIC5, 6; pages 130, 137= Table XV; page 136> U n f o r t u n a t e l y , there was i n s u f f i c i e n t Raia r h i n a m a t e r i a l f o r t h i s f u r t h e r step to be attempted, because the probable l o s s of m a t e r i a l would have e l i m i n a t e d any p o s s i b l e amino a c i d analys i s. Of the n e u t r a l and a c i d i c amino acids present i n the h y d r o l y s a t e , the amino acids which c o n s t i t u t e the g l u m i t o c i n molecule predominated over the o t h e r s , with the exception of methionine. C y s t e i c a c i d , the o x i d a t i o n product of the c y s t e i n e r e s i d u e which occurs i n g l u m i t o c i n i n twice the p r o p o r t i o n of the other amino a c i d c o n s t i t u e n t s , was i n low p r o p o r t i o n . However, c y s t e i n e i s known to break down during h y d r o l y s i s (Chauvet et_ a l . , 1963); analogy with a l l other neurohypophysial p r i n c i p l e s would suggest the presence of two c y s t e i n e r e s i d u e s , and such an assumption i s j u s t i f i e d by the p h armacological p r o p e r t i e s of the skate m a t e r i a l . The amount of methionine i n the h y d r o l y s a t e was s u f f i c i e n t l y high : 1 0 1 (molar r a t i o to glutamic a c i d = 0.89) f o r i t to be considered part of the p e p t i d e . However, methionine had a l s o been present as a contaminant i n the Squalus p r e p a r a t i o n a f t e r i t s e l u t i o n from CM Sephadex (Table XV ); . d e s p i t e i t s presence, i t could be shown that i t could be removed from the p r i n c i p l e by the use of SE Sephadex. T h e r e f o r e , i t i s considered probable t h a t the methionine found here was not part of the p e p t i d e , and that chromatography on SE Sephadex might have separated i t from the B_. r h i n a p r i n c i p l e , i f s u f f i c i e n t m a t e r i a l had been a v a i l a b l e to i n c l u d e t h i s e x t r a step. The e x c l u s i o n of methionine from the a c t i v e peptide i s f u r t h e r supported by the phar m a c o l o g i c a l evidence presented e a r l i e r . The s p e c i f i c a c t i v i t y of the peptide was c a l c u l a t e d from the r a t uterus a c t i v i t y (no Mg + + present) of the hydrolyzed sample and from the number of micromoles of glutamic a c i d i t cont a i n e d ; the value was found to be k.6 IU/mg. This provides a d d i t i o n a l support f o r the suggestion that the peptide might be g l u m i t o c i n . G l u m i t o c i n has a much lower s p e c i f i c a c t i v i t y than the other known n e u t r a l p r i n c i p l e s - 8 IU/mg (Table V I I I , page 88; a l s o Acher e_t_ al_. , 1965) , as compared to U50 IU/mg f o r o x y t o c i n , 290 IU/mg f o r mesotocin (Sawyer, 1965) and 125-150 IU/mg f o r i s o t o c i n (Sawyer, 1965 ; Wilson and Smith, 1969). The a c t i v i t y of the R. r h i n a p r i n c i p l e was i n the same low range as the a c t i v i t y of g l u m i t o c i n . 102 D. Summary of the Studies on the Neurohypophysial  P r i n c i p l e s of Raia r h i n a 1. The p o t e n c i e s of neurointermediate lobe e x t r a c t s of Raia r h i n a were found to vary upon repeated r a t uterus assays, both i n the presence and the absence of magnesium i o n s , and on the m i l k - e j e c t i o n assay. Exact values appeared to depend on the p a r t i c u l a r assay p r e p a r a t i o n used, as w e l l as on the c o n c e n t r a t i o n and nature of the p e p t i d e . T h i s i n d i c -ated the importance of assaying an unknown against a standard of the same molecular s t r u c t u r e whenever i t i s p o s s i b l e . Nevertheless , d e s p i t e the c a u t i o n necessary i n i n t e r p r e t i n g assay v a l u e s , no a l t e r n a t i v e e x i s t s when d e a l i n g with a t o t a l l y unknown p e p t i d e . The e x i s t e n c e of a v a r i a b i l i t y i n assay values obtained f o r an unknown does not d e t r a c t from the v a l i d i t y of t h i s evidence as an i n d i c a t i o n that there i s a d i f f e r e n c e between the unknown and the standard; f u r t h e r , r e p e t i t i v e agreement i n assay values between two peptides i n c r e a s e s the p o s s i b i l i t y that the two are the same, although i t does not prove t h a t t h i s i s the case. 2. Two p r i n c i p l e s , one n e u t r a l and one b a s i c , were separated from the Raia r h i n a e x t r a c t : a. The b a s i c p r i n c i p l e was not p r e c i s e l y c h a r a c t e r i z e d ; however, analogy with other elasmobranchs, i n c l u d i n g skate, and the s i m i l a r i t y of the chromatographic behavior of t h i s a n t i d i u r e t i c m a t e r i a l with t h a t of the a r g i n i n e v a s o t o c i n i d e n t i f i e d i n Squalus, suggested that i t 103 i s a l s o a r g i n i n e v a s o t o c i n . b. The f o l l o w i n g ev idence suggested t h a t the n e u t r a l p r i n c i p l e was g l u m i t o c i n ( I t - ser , 8 - g l u (NH ) o x y t o c i n ) ; i . The r e s u l t s of the p h a r m a c o l o g i c a l com-p a r i s o n between the crude e x t r a c t and s y n t h e t i c g l u m i t o c i n (Tab le VIT,3, page :8'3) were c o n s i s t e n t w i th the p resence of a mix ture of g l u m i t o c i n w i th a r g i n i n e v a s o t o c i n , or wi th any of the known n a t u r a l l y o c c u r r i n g a n t i d i u r e t i c p r i n c i p l e s ; w i th the p r o v i s i o n t h a t the p r o p o r t i o n s i n which the p r i n c i p l e s o c c u r r e d i n the e x t r a c t were the same as those found when the n e u t r a l ( o x y t o c i c ) and b a s i c ( a n t i d i u r e t i c ) f r a c t i o n s were s e p a r a t e d on a CM Sephadex column. i i . The amino a c i d a n a l y s e s of the n e u t r a l p r i n c i p l e (Tab le IX) r e v e a l e d the amino a c i d s of g l u m i t o c i n , p l u s m e t h i o n i n e . The b a s i c amino a c i d s - h i s t a m i n e , l y s i n e , and a r g i n i n e - were a l s o p r e s e n t but were c o n s i d e r e d to be e x t r i n s i c to the a c t i v e m o l e c u l e . Exper iments w i th the p r i n c i p l e s of Squalus a c a n t h i a s suggested t h a t the meth ion ine found here was a c o n t a m i n a n t , and not an i n t r i n s i c par t of the p e p t i d e ; t h i s was suppor ted by the p h a r m a c o l o g i c a l d a t a , s i n c e a s u b s t i t u t i o n or a d d i t i o n of meth ion ine would be expected to d i s t u r b the agreement between the b i o l o g i c a l a c t i v i t i e s of the e x t r a c t and those o f s y n t h e t i c g l u m i t o c i n . i i i . The s p e c i f i c a c t i v i t y of the n e u t r a l . p r i n c i p l e was low , and comparable to tha t of g l u m i t o c i n . 10k I I . STUDIES OF THE NEUROHYPOPHYSIAL PRINCIPLES OF THE DOGFISH, SQUALUS ACANTHIAS, (PACIFIC VARIETY) A. I n t r o d u c t i o n This species had already "been s t u d i e d by other workers , but with c o n f l i c t i n g r e s u l t s . H e l l e r and P i c k e r i n g (1961) had r e s o l v e d a mixture of the two o x y t o c i c p r i n c i p l e s , E^ and E 2 5 by paper chromatography of p i t u i t a r y e x t r a c t s of the A t l a n t i c v a r i e t y of t h i s s p e c i e s . In c o n t r a s t , Perks (1966) had been unable to d i s t i n g u i s h more than one a c t i v e f r a c t i o n , EOP I, when u s i n g the same method, and Sawyer (1965) had r e s o l v e d only one o x y t o c i n - l i k e f r a c t i o n by ion exchange chromatography of neurohypophysial m a t e r i a l from the P a c i f i c v a r i e t y of t h i s s p e c i e s . An attempt was made to e x p l a i n t h i s c o n f l i c t and to e l a b o r a t e on the nature of the p r i n c i p l e or p r i n c i p l e s produced by t h i s s p e c i e s . T h i s was done by pharmacological and chromatographic s t u d i e s of crude e x t r a c t s , and by the p r e p a r a t i o n of p u r i f i e d p r i n c i p l e s which could then be sub-j e c t e d to more r i g o r o u s p h a r m a c o l o g i c a l , chromatographic and chemical examination. The s y n t h e s i s of g l u m i t o c i n , a known elasmobranch p r i n c i p l e , d u r i n g the course of t h i s study (Manning et a l . , 1968) p e r m i t t e d the d i r e c t p h armacological comparison of the Squalus p r i n c i p l e a gainst a peptide of p o s s i b l y i d e n t i c a l s t r u c t u r e . The d o g f i s h was of a d d i t i o n a l i n t e r e s t because i t provided a c o n t r a s t with the skate, Raia r h i n a , the other species s t u d i e d here. Squalus acanthias i s a shark, and i t 1 0 5 has a d i s c r e t e neurohypophysial s t r u c t u r e ( M e u r l i n g , 1 9 6 2 ) as opposed to the d i f f u s e type found among the species of Raia ( M e u r l i n g , 1 9 6 7 a ) . An attempt was made to determine whether these d i f f e r e n c e s between the two species would be p a r a l l e l e d by d i f f e r e n c e s i n t h e i r neurohypophysial p e p t i d e s . B. Ph armacological and Chromatographic Studies of the  Neurointermediate Lobe E x t r a c t s 1 . B i o l o g i c a l A c t i v i t i e s and A c t i v i t y Ratios of the  Crude E x t r a c t s S e v e r a l crude e x t r a c t s were assayed a g a i n s t s y n t h e t i c o x y t o c i n (Syntocinon, Sandoz Pharmaceuticals) f o r r a t uterus a c t i v i t y , both i n the presence and absence of magnesium i o n s , and f o r m i l k - e j e c t i o n a c t i v i t y . They were assayed against v a s o p r e s s i n ( P i t r e s s i n , Parke Davis) f o r a n t i d i u r e t i c a c t i v i t y . The r a t i o s of t h e i r a c t i v i t i e s were compared to those of other elasmobranchs. The r e s u l t s , shown i n Table X, were comparable to those found f o r t h i s s p e c i es by other workers (Sawyer e_t_ ajL. , 1 9 6 l ; Perks and Dodd, 1 9 6 3 a j Sawyer, 1 9 6 5 ; 1 9 6 7 ; S w i a t k i e w i c z , 1 9 6 8 ) . The l e v e l s of o x y t o c i c a c t i v i t y ranged from 1 1 . 6 to 3 2 . 2 mU/mg d r i e d t i s s u e . These were higher than the l e v e l s found f o r Raia r h i n a , the other species s t u d i e d here, but were comparable to the l e v e l s of a c t i v i t y r e p o r t e d f o r s e v e r a l other elasmobranch species (Perks and Dodd, 1 9 6 3 a ) . The a c t i v i t y r a t i o s were also s i m i l a r to those of other elasmobranchs (Perks and Dodd, 1 9 6 3 a ) . The r a t i o of a n t i d i u r e t i c to r a t uterus a c t i v i t y was t y p i c a l l y low ( RADH = 0*03 or 0 . 0 4 ) and suggested that an a n t i d i u r e t i c -Table X The A c t i v i t i e s and A c t i v i t y Ratios of Crude Neurointermediate Lobe E x t r a c t s of Squalus acanthi as Values found i n t h i s study Sample number 3 A c t i v i t i e s * 3 (mU/mg d r i e d t i s s u e ) A c t i v i t y r a t i o s RU C ME ADH RU+Mg R ME RADH Mg 27-39 ( i n c l ) (998 NIL) 25.0 ± 2.6 55.1 ± 13.0 •O.TO ± 0.29 51. h ± 10.1 2.2 ± 0 . 5 0.03 ± 0.01 2.1 ± 0. 5 25 (21 NIL) 12.3 ± 0.7 - 0.1+8 ± 0.15 55.2 ± 16.8 - 0.01+ ± 0.01 ± 1. 1+ • 26 (26 NIL) 11. 6 ± 1.3 0.1+5 ± 0.22 3l+. 2 ± 13. 1+ - 0.0U ± 0. 02 2.9 ± 1. 1 36 (5 NIL) 32.2 ± 5 - 0 - - 63.8 ± 12.0 - - 2.0 ± 0. 5' Table X continued Values found by other workers L o c a t i o n Rat uterus a c t i v i t y (mU/mg d r i e d t i s s u e ) A c t i v i t y r a t i o s Reference RME R A D H RMg B r i t i s h Columbia - 1 . 2 - 3 . 2 S w i a t k i e w i c z , 1968 Washington State - 1 . 6 e 0 . 0 2 e 1.6±0. 1 Sawyer, 1965 Maine 30 ±h • 1 . 9 e 0.03 e 1 . 3 e Sawyer et_ a l , , 1961 V i r g i n i a - - 0. 06 2 . 6 ± 0 . 1 Sawyer, 1967 Scotland 5 7 . 0 ± 3 . 8 - 0 . 09 - Perks and Dodd ,1963 S c o t l a n d 17 2.6 0. 07 - Perks and Dodd ,1963 Sc o t l a n d 35 ± 3 . 5 1 . 8 e 0. 0 2 e 2. 0 e Sawyer _et_ al_. , 1961 As i n Appendix I. Assays c a l c u l a t e d by the U-point method of Holton (19^8); whenever three or more groups were obtained the f i d u c i a l l i m i t s are expressed, P = 0 .05 . c A b b r e v i a t i o n s used: RU = a c t i v i t y on r a t u t e r u s , i n absence of magnesium. ME = a c t i v i t y on r a b b i t m i l k - e j e c t i o n assay. ADH = a c t i v i t y on r a t a n t i d i u r e t i c assay. RU+Mg = a c t i v i t y on r a t u t e r u s , i n presence of 0.5 mM Mg + +. A c t i v i t y r a t i o s , R M E, RADH » a n d - RMg» a r e "kke r a t i o s of the a c t i v i t y found on each assay (ME, ADH, and RU+Mg) to the a c t i v i t y found on the rat uterus assay, without magnesium (RU). d A l s o l i s t e d i n Table XII. e C a l c u l a t e d from the p u b l i s h e d data. p r e s s o r p r i n c i p l e was e i t h e r absent or present i n very s m a l l amounts. The r a t i o of m i l k - e j e c t i o n to r a t uterus a c t i v i t y (R,„ = 2.2 + 0.5) c h a r a c t e r i z e d the o x y t o c i c f r a c t i o n as mill EOP I, a c c o r d i n g to the d e f i n i t i o n of Sawyer (1964). This p r i n c i p l e has been r e p o r t e d i n seven other species (Perks and Dodd, 1963a; Perks, 1966) and i s d i s t i n g u i s h e d from EOP II by p o s s e s s i n g a lower value f o r R . Values have not been r e p o r t e d f o r the magnesium p o t e n t i a t i o n of the EOP I produced by these other s p e c i e s , but the values found here f o r Squalus acanthias (Rjyjg = 2.0 to 4.5) are comparable to those found p r e v i o u s l y f o r the same s p e c i e s . T h i s p o t e n t i a t i o n does not d i s t i n g u i s h the Squalus p r i n c i p l e from e i t h e r Eg ( = 2) or from EOP II (R„ = 2.6) but i t does d i f f e r e n t i a t e i t from Mg the E^ p r i n c i p l e found i n c e r t a i n species (Rj^g = 6) and from g l u m i t o c i n (R^ g = 10). However, i n view of the o b s e r v a t i o n s made on the v a r i a b i l i t y of magnesium p o t e n t i a t i o n s ( S e c t i o n IB2, page 71) t h i s c h a r a c t e r i s t i c must be viewed with c o n s i d e r a b l e s c e p t i c i s m . 2. Studies of A c e t o n e - E x t r a c t a b l e B i o l o g i c a l  Ac t i v i t i e s A p o s s i b l e reason why the two n e u t r a l p r i n c i p l e s , and Egj which were found i n t h i s species by H e l l e r and P i c k e r i n g (1961), could not be d i s t i n g u i s h e d by e i t h e r Sawyer (1965) or Perks (1966) might have been the l o s s of one of the peptides i n t o the acetone i n which the glands were c o l l e c t e d . The p r e f e r e n t i a l e x t r a c t i o n of one peptide (oxytocin) over 109 another ( v a s o p r e s s i n ) had been observed by H e l l e r and L e d e r i s (1959) when the p o s t e r i o r p i t u i t a r y glands from i n f a n t r a t s were c o l l e c t e d i n acetone. Therefore the acetone i n t o which the glands of samples 35 and 36 (Appendix I) were d i s s e c t e d , and the two changes of acetone i n which they stood (at approximately 12°C) f o r the f i r s t hour a f t e r d i s s e c t i o n were combined f o r each sample. A t o t a l of 250 ml of acetone e x t r a c t was obtained from each set of glands. The acetone i n which the glands were s t o r e d (at approximately 12°C) between 72 and ihk hours a f t e r d i s s e c t i o n was al s o kept. The four samples were f l a s h evaporated to the p o i n t of dryness. The r e s i d u e s were taken up i n 0.25% a c e t i c a c i d s o l u t i o n and assayed on the r a t uterus f o r o x y t o c i c a c t i v i t y . The r e s u l t s , l i s t e d i n Table XI, show that the acetone i n which the glands stood fo r the f i r s t hour a f t e r d i s s e c t i o n contained a c t i v i t y , U88 ± 31 mU r a t uterus a c t i v i t y (without Mg + +) from sample 35 and 706 ± 102 mU r a t uterus a c t i v i t y (without Mg + +) from sample 36, but that no a c t i v i t y was l o s t from the glands between 72 and ikk hours a f t e r d i s s e c t i o n . The a c e t o n e - e x t r a c t a b l e a c t i v i t y was i n a c t i v a t e d by i n c u b a t i o n with sodium t h i o g l y c o l l a t e . T h i s i n d i c a t e d that i t was i n f a c t due to the presence of neurohypophysial hormone r a t h e r than to some other o x y t o c i c f a c t o r which might have been leached from the glands. At l e a s t Q0% and 85% of the Table XI The Oxytocic A c t i v i t y E x t r a c t e d from Two Samples of Squalus acanthias Neurointermediate Lobes by the Acetone Used f o r C o l l e c t i o n Sample number2 Number of glands Storage i n acetone Hours a f t e r Volume of d i s s e c t i o n acetone (ml) Rat uterus a c t i v i t y i n the acetone T o t a l a c t v i t y % of t o t a l (mU) a c t i v i t y i n glands I n a c t i v a t i o n by sodium t h i o g l y c o l l a t e 35 50 0-1 7 2 - l l A 250 50 488 ± 31 none >80% 36 79 0-1 12-lhk 250 36 706 ± 102 none 3.7 >85% As i n Appendix I. I l l a c t i v i t y l o s t from samples 35 and. 36, r e s p e c t i v e l y , was destroyed upon k 1/2 and 1 1/2 hours exposure to sodium t h i o g l y c o l l a t e (Table XI). In sample 36, i t was p o s s i b l e to show that the a c t i v i t y r e covered from the acetone accounted f o r only a small p r o p o r t i o n of that present i n the glands - only about k% of the t o t a l a c t i v i t y i n the sample. F i v e glands from t h i s sample were e x t r a c t e d i n 0.25% a c e t i c a c i d and found to have a r a t uterus a c t i v i t y of 30.1 ± 2.9 mU/mg d r i e d t i s s u e . This was e x t r a p o l a t e d to an a c t i v i t y of 18,1+76 mU f o r the t o t a l sample of 613.2 mg d r i e d t i s s u e (Appendix I ) . The 706 mU recovered from the acetone accounted f o r 3.7% of the t o t a l (Table X I ) . The a c e t i c a c i d e x t r a c t of sample 36 and the a c t i v -i t y recovered from i t s " f i r s t hour" acetone were compared f o r magnesium p o t e n t i a t i o n and f o r chromatographic behavior. These two c r i t e r i a d i s t i n g u i s h p r i n c i p l e E^ from E^. The two unknowns were assayed side by side on the same r a t u t e r i , both with and without magnesium ions present. T h e i r r e s u l t i n g magnesium p o t e n t i a t i o n s were s i m i l a r , 2.0 ± 0.5 f o r the standard e x t r a c t of the glands, and 2.1 ± 0 . 5 f o r the acetone s o l u b l e r e s i d u e (Table X I I ) ; n e i t h e r approximated to the high values r e p o r t e d f o r p r i n c i p l e E^ i n some species ( H e l l e r and P i c k e r i n g , 1 96l; H e l l e r and Roy, 1965a; 1965b; Roy, 1969). The e x t r a c t and the acetone s o l u b l e r e s i d u e were a p p l i e d side 112.., Table XII The Magnesium P o t e n t i a t i o n and Chromatographic Behavior of a Crude E x t r a c t of Squalus acanthias Neurointermediate Lobes, and of the A c t i v i t y Recovered from the Acetone i n which the Glands were C o l l e c t e d Source of Magne s ium Chromatographic a c t i v i t y p o t e n t i a t i o n b e h a v i o r 8 . ( R M g > (R F) A c e t i c a c i d • extract* 3 2.0 ± 0.5 C 0.1+ - 0.7 Acetone s o l u b l e re s i due 2.1 ± 0.5 0.1+ - 0.8 Solvent = n - b u t a n o l : a c e t i c acid:water, 4:1:5. Crude e x t r a c t of f i v e glands from Sample 36 (Appendix I ) . c A l s o l i s t e d i n Table X. ^•Residue recovered from the 250 ml of acetone i n t o which the glands of Sample 36 were d i s s e c t e d and s t o r e d f o r one hour. 113 by side to a paper chromatogram which was then developed i n n - b u t a n o l : a c e t i c acid:water = 4:1:5 - the system used by H e l l e r and P i c k e r i n g (1961) to separate E^ from Eg. Oxytocic a c t i v i t y was found i n the eluates of R„ 0 .4-0.7 i n the case r of the e x t r a c t , and R^ 0 .4-0.8 f o r the acetone s o l u b l e r e s i d u e (Table XII; F i g . 5 9 b and c ) . No a c t i v i t y was found f o r e i t h e r the e x t r a c t or the r e s i d u e i n the eluates of B.^ F 0-0 . 4 , where E^ would be expected to run. Nor was there evidence f o r the incomplete s e p a r a t i o n of E^ from Eg. The p o t e n t i a t i o n of the t a i l i n g edge of the o x y t o c i c peak, R if 0 .4-0.5» was 2.3 f o r the e x t r a c t and 1.8 f o r the acetone s o l u b l e r e s i d u e . These values were s i m i l a r i n each case to the o v e r a l l p o t e n t i a t i o n of 2.0 f o r the t o t a l a c t i v e eluate of the e x t r a c t and 2 .4 f o r the t o t a l a c t i v e e l u a t e of the acetone s o l u b l e r e s i d u e . T h e r e f o r e , although approximately h% of the a c t i v i t y of the glands was l o s t i n t o the c o l l e c t i n g acetone, t h i s a c t i v i t y appeared s i m i l a r to the p r i n c i p l e e x t r a c t e d l a t e r by a c e t i c a c i d ; and there was no evidence f o r the presence of the slow running, and sometimes h i g h l y p o t e n t i a t e d , p r i n c i p l e E^ i n e i t h e r f r a c t i o n . C. The P u r i f i c a t i o n of the E x t r a c t An attempt was made to p u r i f y the neurohypophysial p r i n c i p l e s produced by t h i s species so that they could be subjected to more r i g o r o u s pharmacological and chromato-graphic examination, and to chemical a n a l y s i s . The general 114 F i g u r e 5. Descending paper chromatograms of Squalus  acanthias neurointermediate lobe e x t r a c t , at v a r i o u s stages of p u r i f i c a t i o n , on Whatman 3MM paper, i n n-butanol: acet i c a c i d : water = 4:1:5 9 at room temperature. Samples used; a: s y n t h e t i c o x y t o c i n (Syntocinon, Sandoz), b: crude e x t r a c t of f i v e glands from Sample 36 ( see^Appendioc I,), c : r e s i d u e recovered from the acetone i n t o which the glands of Sample 36 were d i s s e c t e d and • s t o r e d f o r one hour, d: e x t r a c t a f t e r p a r t i a l p u r i f i c a t i o n on Sephadex G-15 (Pre-p a r a t i o n 2) , e: o x y t o c i c f r a c t i o n 1 (Pre-p a r a t i o n 2 ) , e l u t e d from CM Sephadex, f : o x y t o c i c f r a c t i o n 2 ( P r e p a r a t i o n 2 ) , e l u t e d from CM Sephadex. Loads are s t a t e d as mU o x y t o c i c a c t i v i t y =- mU r a t uterus a c t i v i t y i n the absence of Mg + +. 115 c o tn > •6 u. CE 0) c o E o •D B U O o > 8 E o o T 30 O ~ 4 0 c « V) .O 1 A cn 2 3 0 0 b Squalus, c r u d e e x t r a c t A c e t o n e so lub le r e s i d u e 1 0 £ 1 0 V) 4) L. a E 0 3 1 0 8 c cn o 2 0 Sephadex G - 1 5 e l u a t e C M S e p h a d e x e l u a t e , f r a c t i o n 1 f C M Sephadex e l u a t e , f r a c t i o n 2 L O A D RECOVERY Oxytocic activity (mU) 1 0 0 1 0 2 6 0 1 0 6 7 3 3 3 0 42°/» 6 6 % 4 2 % , 5 4 ° / o 1 2 - 3 .4 - 5 - 6 - 7 R F •8 - 9 1 0 116 p u r i f i c a t i o n scheme, which i s o u t l i n e d i n F i g u r e 6a, i n c l u d e d g e l f i l t r a t i o n of t h e e x t r a c t , f o l l o w e d by t h r e e i o n exchange chromatography p r o c e d u r e s , and a f i n a l p u r i f i c a t i o n by paper chromatography. The procedures were c o m p l i c a t e d by the need t o combine f r a c t i o n s from d i f f e r e n t p u r i f i c a t i o n s i n o r d e r t o conserve enough m a t e r i a l t o a l l o w f o r amino a c i d a n a l y s i s . T h i s i s shown i n t h e more d e t a i l e d o u t l i n e of the p u r i f i c a t i o n scheme p r e s e n t e d i n F i g u r e 6b. At v a r i o u s stages i n t h e i r p u r i f i c a t i o n , as i n d i c a t e d i n the two f i g u r e s , the p r i n c i p l e s were s u b j e c t e d t o p h a r m a c o l o g i c a l or chromatographic s t u d i e s , or t o h y d r o l y s i s and amino a c i d a n a l y s i s . 1. E x t r a c t i o n of the Glands With the e x c e p t i o n o f the f i v e glands which were used i n t h e p r e v i o u s experiment ( S e c t i o n B 2 , page 108) the glands of samples 27-39 (Appendix I ) were t a k e n f o r p u r i f i c a t i o n . The 998 a c e t o n e - d r i e d g l a n d s , w e i g h i n g 6500 mg, were e x t r a c t e d i n 0.25% a c e t i c a c i d at ho mg d r i e d t i s s u e / m l . The r a t .uterus a c t i v i t y ( w i t h o u t Mg ) of the crude e x t r a c t was 998 ± 105 mU/ml, or 25.0 ± 2.6 mU/mg d r i e d t i s s u e (Table X ) . I t ' s Lowry p e p t i d e c o n t e n t was 7.95 mg/ml and the r a t i o of o x y t o c i c a c t i v i t y t o Lowry p e p t i d e was 125.5 mU/mg. The e x t r a c t was s t o r e d f r o z e n u n t i l r e q u i r e d . Wo l o s s of a c t i v i t y was i n c u r -r e d , even a f t e r s t o r a g e of up t o 15 months. 2. P r e l i m i n a r y P u r i f i c a t i o n on Sephadex G-15 A. The e x t e n t of p u r i f i c a t i o n The a c t i v e p e p t i d e s were s e p a r a t e d away from the l a r g e r 117 Figure 6a. General o u t l i n e of the procedures f o l l o w e d during the p u r i f i c a t i o n of the a c t i v e p r i n -c i p l e s from the neurointermediate lobe (NIL) °^ Squalus acanthias ( P a c i f i c v a r i e t y ) . . 118 I n e u t r a l Fraction 1 9 9 8 Squalus NIL 0 . 2 5 ° / o acetic acid 133 ml crude extract Sephadex G-15 (Ion retardation resin) Flash evaporation DEAE Sephadex CM Sephadex •paper chromatogram fractions I Fraction 2 basic fraction Freeze drying SE Sephadex Freeze drying CM Sephadex Fraction 3 \ paper chromatogram isoelectric focusing Freeze amino acid analysis drying SE Sephadex —--—--—--v f~ amino acid analysis combined neutral fractions Freeze drying Paper chromatography — isoelectric focusing amino acid analysis — amino acid analysis Figure 6a. General outline of the procedures followed during the purification of the active principles from the neurointermediate lobe (NIL) of Squalus acanthias ( Pacific variety ). 11 Si-F i g u r e 6t>. D e t a i l e d o u t l i n e of the procedures, followed during the p u r i f i c a t i o n of the a c t i v e p r i n -c i p l e s from the neurointermediate lobe (NIL) o f Squalus acanthias ( P a c i f i c v a r i e t y ) . 998 Squalus NIL (6500 mg acetone dried tissue) 0.25V. acetic acid I 133ml crude extract (132-7 IU oxytocic activity) 9-9ml Preparation 1 Flash evaporation DEAE Sephadex CM Sephadex trial elution Preparation 2 Flash evaporation neutral fraction basic fraction r DEAE Sephadex Flash evaporation CM Sephadex Paper chromatogram elution pattern 1 Preparation 3 Flash evaporation DEAE Sephadex CM Sephadex elution pattern 1 T neutral fractions Fraction 1 Fraction 2 1 Preparation 4 Ion retardation resin Flash evaporation DEAE Sephadex CM Sephadex elution pattern 2 basic neutral fract ons basic neutral fractions fraction I | fraction! I I I I I I I I Fraction 3 Fractionl Fraction2 Fraction3 Fractionl Fraction2 Fraction 3 I I I biological assays "I basic fraction 1 paper FD chromatogram FD I IFD paper chromatogram amino biological II analysis assays V neutral fraction lb SE Sephadex amino acid analysis neutral isoelectric focusing FD fraction la neutral fraction 2a biological | assays amino acid analysis F D neutral fraction lc neutral SE Sephadex amino acid analysis SE Sephadex amino acid analysis (Table 24 ) biological assays SE Sephadex FD CM Sephadex elution pattern 2 FD fraction 2b SE Sephadex biological assays FD - freeze drying Figure 6b Detailed outline of the procedures followed during the purification of the active principles from the neurointermediate lobe (NIL) of Squalus acanthias ( Pacific variety). combined neutral fraction amino isoelectric acid focusing Paper chroma-analysis tography (Table 24) amino acid analysis ( Table 24 ) 121 p r o t e i n molecules i n the e x t r a c t by passage of the e x t r a c t through a 90 cm x 2 .4 cm column of Sephadex G-15. This was the f i r s t time a Sephadex g e l with such a low e x c l u s i o n l i m i t (molecular weight of approximately 1500) had been used i n the p u r i f i c a t i o n of elasmobranch p r i n c i p l e s . The higher degree of p u r i f i c a t i o n a f f o r d e d by t h i s g e l was e s p e c i a l l y advantageous to elasmobranch e x t r a c t s , which have such a low l e v e l of o x y t o c i c a c t i v i t y (Perks and Dodd, 1963a; Perks, 1966). The e x t r a c t was passed through the column i n ten p o r t i o n s of 10 ml each. F i g u r e 7 shows the s e p a r a t i o n of the a c t i v e p r i n c i p l e s from the l a r g e r p r o t e i n s i n the e l u a t e . The r e s u l t s of each run are summarized i n Table X I I I . An average of 92% of the Lowry peptide was separated away from the o x y t o c i c p r i n c i p l e s , and the r a t i o of o x y t o c i c a c t i v i t y to Lowry peptide was i n c r e a s e d approximately 1 3 - f o l d . The e x t r a c t was d i l u t e d 9 - f o l d by t h i s procedure. In an attempt to decrease the d i l u t i o n , the remaining 34.4 ml of e x t r a c t was a p p l i e d to the column i n one l o t . The s e p a r a t i o n of the a c t i v e peptides from the l a r g e r p r o t e i n molecules was not as complete as when the samples were a p p l i e d i n 10 ml amounts, but 86% of the Lowry peptide was removed and a 6.2-fold p u r i f i c a t i o n was achieved. The r e d u c t i o n i n p u r i f i c a t i o n was o f f s e t by a 50% r e d u c t i o n in:, "the d i l u t i o n of the p r i n c i p l e s . 122 Figure 7. Chromatogram of 10 ml of crude Squalus acanthias neurointermediate lobe e x t r a c t on a 90 cm x 2.U cm column of Sephadex G-15. Empty t r i a n g l e s = Lowry peptide content of the e l u a t e . Empty c i r c l e s = o x y t o c i c a c t i v -i t y ( r a t uterus a c t i v i t y , without Mg + +) of the e l u a t e . 123 Squalus acanthias, chromatography on Sephadex G-15 Eluate volume (ml) H 3" 3 o 3 13 O O 5' o on o c o E. £' g-5 to vo o On K-* 1 I OJ OJ 4^ 4^ o o J i . OJ On vo 4* 4* 'h-- ON IO ^ 1 oo to oo X £ o o o o OJ 4^ to o I I I I to to h-. to o ~ o ~ I—' O OJ to VO 00 OJ 4* OJ to to to to o i j ^ . to - j OJ 4^ - On i—» I I I I OJ OJ OJ OJ on to 00 4i. 4* on 4^ to <] oo OJ 00 o\ 0J vo o o ON to X 00 o O bo O O vo VO B P S 03 vo vo to o i to to to ON On O l i—» OJ on OJ OJ OJ o i OJ 4^ Ji. ~ j on on o to to to ON on 4^ ^ to VO I I I OJ OJ OJ on J> OJ i— 4*. vo VO 00 00 00 to oo OJ OJ vo oo po po to bo OJ OJ vo 00 VO VO VO VO OJ 4^ o o to o O po vo ON j> o O tO I— X to oo VO to VO on 4^ VO 4* O o ON X O c a-a X a o -n to Experiment number Sample volume (ml) Position o f minor oxytocic peak in the eluate (cumulative volume, ml) Position of major oxytocic peak in the eluate (cumulative volume, ml) Volumesof the major peak (ml) Dilution o f activity in major peak "Preparation" number assigned to the combined major peaks Specific activity - ratio of oxytocic acitivty to L o w r y peptide (mU/mg) % L o w r y peptide removed from crude extract % Recovery o f oxytocic activity Degree of purification 125 This was an advantage i n view of the already low l e v e l of o x y t o c i c a c t i v i t y i n the e x t r a c t . The major o x y t o c i c f r a c t i o n s (see Table XIII) e l u t e d from runs 1-3 were combined as " P r e p a r a t i o n l " , and from runs k-6 and 7-10 as " P r e p a r a t i o n 2" and " P r e p a r a t i o n 3", respec-t i v e l y . The a c t i v e e l u a t e from the eleventh run became " P r e p a r a t i o n V . Each was processed s e p a r a t e l y . b. The minor o x y t o c i c peak A small amount of o x y t o c i c a c t i v i t y ( l . k % of the t o t a l ) was e l u t e d from the column along with the Lowry peptide and i n advance of the main o x y t o c i c peak ( F i g . J, Table X I I I ) . T h i s might have represented som o x y t o c i c agent other than a neurohypophysial p r i n c i p l e . However when two samples of t h i s p r e l i m i n a r y a c t i v i t y were incubated f o r s e v e r a l hours with sodium t h i o g l y c o l l a t e , they were completely i n a c t i v a t e d . Therefore t h i s minor peak probably represented neurohypophysial peptide which may s t i l l have been i n a s s o c i a t i o n with the c a r r i e r p r o t e i n . Such a phenomenon has been d e s c r i b e d f o r the passage of p i t u i t a r y e x t r a c t s of the cobra, Naj a naj a, through Sephadex G-25 ( P i c k e r i n g , 1967). c . Paper chromatography of the Sephadex' G-15 e l u a t e Trace amounts of a b a s i c p r i n c i p l e , which was pharmac-o l o g i c a l l y s i m i l a r to a r g i n i n e v a s o t o c i n , had been separated away from the main n e u t r a l f r a c t i o n of Squalus acanthi as by i o n exchange chromatography (Sawyer, 1965). However i t had 126 not been detected i n paper chromatograms of crude e x t r a c t s of the same species (Perks, 1°66), p o s s i b l y because the danger of o v e r l o a d i n g the paper had l i m i t e d the amount of e x t r a c t which was a p p l i e d . An attempt was made here to demonstrate the b a s i c pep-t i d e by paper chromatography, t h i s time using p a r t i a l l y p u r i f i e d e x t r a c t so that higher loads of a c t i v i t y could be a p p l i e d . A 3.4'ml sample of the Sephadex G-15 eluate ( P r e p a r a t i o n 2) con-t a i n i n g 1067 mU o x y t o c i c a c t i v i t y was reduced by f l a s h evapor-a t i o n to a volume of 0.3 ml, with no l o s s of a c t i v i t y . I t was a p p l i e d to a paper chromatogram beside an a r g i n i n e v a s o t o c i n standard. A f t e r the chromatogram had been developed i n n-b u t a n o l : a c e t i c acid:water = 4:1:5» the s y n t h e t i c a r g i n i n e vaso-t o c i n was l o c a t e d by r a t uterus assay i n the e l u a t e of R„ 0.3-0.4. A t o t a l of 577 mU of o x y t o c i c a c t i v i t y was recovered from the chromatogram of the Squalus e x t r a c t , mainly i n the e l u a t e s of R„ 0.4-0.7 ( F i g . 8). However a t r a c e amount of o x y t o c i c a c t i v i t y (4.5 mU) occurred i n the e l u a t e of R„ 0.3-0.4. This r e l u a t e a l s o contained most of the a n t i d i u r e t i c and f r o g bladder a c t i v i t y recovered from the paper (12 mU and 2136 mU, respec-t i v e l y ) and i t s a c t i v i t y r a t i o s , R.^^ = 2.7 and R = 475, were comparable to the values of RftT>TJ = 2.0 and R = 271-910 re p o r t e d f o r a r g i n i n e v a s o t o c i n (Sawyer e_t al_. , 1961 ; Sawyer, 1965). Such a high r a t i o of f r o g bladder to r a t uterus a c t i v i t y i s a p e c u l i a r c h a r a c t e r i s t i c of a r g i n i n e v a s o t o c i n ; the only other o x y t o c i n analogue known to have an R at a l l 127 F i g u r e 8. Descending paper chromatogram of Squalus . acanthias neurointermediate lobe e x t r a c t ( P r e p a r a t i o n 2, a f t e r p a r t i a l p u r i f i c a t i o n on Sephadex G-15) on Whatman 3MM paper, i n n - b u t a n o l : a c e t i c acid:water = 4:1:5, at room temperature. The three diagrams f o r the Squalus e x t r a c t show the d i s t r i b u t i o n of the t h r ee a c t i v i t i e s : r a t uterus a c t i v i t y , without Mg + + ( a l s o shown i n Fi g u r e 5d); r a t a n t i d i u r e t i c a c t i v i t y ; and f r o g bladder a c t i v i t y . Loads are s t a t e d as mU o x y t o c i c a c t i v i t y = mU r a t uterus a c t i v i t y i n the ab-sence of Mg + +. 12 8" c o > •5 u. or <u c O E o i_ T3 0) -t-> > <J o z> E o .o 8 0 0 8 0 30?) 0 1 5 O 2 5 0 0 S y n t o c i n o n A r g i n i n e vasotoc in Squalus o x y t o c i c (rat uterus) a c t i v i t y a n t i d i u r e t i c a c t i v i t y f r o g b ladder a c t i v i t y LOAD Oxytocic activity (mU) 200 230 1067 RECOVERY 29%, 32°/. 54% •1 - 2 .4 - 5 R F •8 9 L O 129 comparable to that of a r g i n i n e v a s o t o c i n i s 8 - l y s i n e o x y t o c i n (R,-,_ = 190; Sawyer, 1965). However the 8 - l y s i n e peptide r B d i f f e r s from a r g i n i n e v a s o t o c i n i n having a low of 0.31, A D n r a t h e r than a value of R ^ - Q J J = 2. 3 . Passage of the .Extract through Ion R e t a r d a t i o n  Resin The s a l t content of the e x t r a c t s n e c e s s i t a t e d a high d i l u t i o n before the p r i n c i p l e s could be adsorbed onto the subsequent c a t i o n exchangers. Therefore i n the p u r i f i c a t i o n of P r e p a r a t i o n k a procedure was i n t r o d u c e d f o r the removal of s a l t from the s o l u t i o n . The a c t i v e e l u a t e from the Sephadex G-15 column was passed through a kO cm x 0.9 cm column of Bior a d ag11a8 Ion R e t a r d a t i o n Resin. The s p e c i f i c c o n d u c t i v i t y of the s o l u t i o n , which a l s o contained the 0.2 M a c e t i c a c i d eluant of the Sephadex G-15 ( s p e c i f i c c o n d u c t i v i t y 0.71 mmho/cm) was reduced from 1.23 mmho/cm to 0.9^ mmho/cm by t h i s procedure. No l o s s of o x y t o c i c a c t i v i t y was detec t e d . h. Passage of the E x t r a c t through DEAE Sephadex Although the neurohypophysial peptides are not them-selve s adsorbed onto DEAE Sephadex, other p r o t e i n s or pep-t i d e s i n the sample do become attached to the g e l , and so are separated away from the a c t i v e p r i n c i p l e s . This step had been found necessary f o r the subsequent a d s o r p t i o n of the ox y t o c i c p r i n c i p l e from the skate, Raia o c e l l a t a , onto the c a t i o n exchanger, CM Sephadex (Perks and Sawyer, 1 9 6 5 ) 9 and so i t was adopted here. 130 Each p r e p a r a t i o n was adjust e d to pH 7-0 and s p e c i f i c c o n d u c t i v i t y 0.21 mmho/cm by dropwise a d d i t i o n of WH^OH and d i l u t i o n with water. Each was passed through a separate 35 cm x 0.9 cm column of DEAE Sephadex which had been e q u i l i b -r a t e d with 0.002 M NH^aeetate - ( PH 7.0). There was no l o s s of o x y t o c i c a c t i v i t y from the samples during t h e i r passage through t h i s exchanger. The Lowry peptide content of the el u a t e could be detected only i n the case of Pr e p a r a t i o n 2, where i t was found that 19% of the Lowry peptide i n the sample had been removed by t h i s procedure. The r a t i o of o x y t o c i c a c t i v i t y to Lowry peptide content had i n c r e a s e d from 1,218 mU/mg to 1,758 mU/mg, a 1 . 4 f o l d p u r i f i c a t i o n of the a c t i v e p r i n c i p l e s . 5. Chromatography on CM Sephadex a. The passage through the column The e l u a t e from each DEAE Sephadex column was adjusted to pH 5.0 and s p e c i f i c c o n d u c t i v i t y 0.l6 mmho/cm by dropwise a d d i t i o n of a c e t i c a c i d and d i l u t i o n with water. Each prepar-a t i o n was a p p l i e d to a separate 35 cm x 0.9 cm column of CM Sephadex, which had been e q u i l i b r a t e d with 0.002 M NH^-ac et at e (pH 5.0). The neurohypophysial peptides were adsorbed onto t h i s exchanger, but could be e l u t e d o f f on an T vM^ acetate g r a d i e n t of i n c r e a s i n g c o n c e n t r a t i o n . P r e p a r a t i o n 1 was used to estab-l i s h the c o n c e n t r a t i o n s of b u f f e r which were necessary to e l u t e 131 the p r i n c i p l e s . The e l u a t e c o n t a i n i n g the n e u t r a l f r a c t i o n was not used f u r t h e r i n the p r e s e n t e x p e r i m e n t s . I t was l y o p h i l i z e d and sent t o Dr. W. Sawyer, Columbia U n i v e r s i t y , f o r f u r t h e r s t u d y . H i s f i n d i n g s are c o n s i d e r e d i n the D i s c u s s i o n , Chapter h. The e l u t i o n p a t t e r n s from the o t h e r t h r e e columns ( P r e p a r a t i o n s 2, 3, and h) were a l l s i m i l a r ( f o r example, F i g . 9 ) . Two n e u t r a l peaks appeared on the g r a d i e n t between 0.002 M NH^acetate- (pH 5.0) and e i t h e r 0.05 M (pH 5 . 0 ) , i n the case of P r e p a r a t i o n s 2 and 3 (CM Sephadex Lot T06071) 5 or 0.10 M (pH 5-0) i n the case of P r e p a r a t i o n k (CM* Sephadex Lot 8852). A t h i r d , b a s i c , peak was e l u t e d by 0.20 M NH^-a c e t a t e (pH 7-0). Table XIV shows the amounts of a c t i v i t y i n each peak . b. The c h e m i c a l a n a l y s i s o f the two n e u t r a l peaks The appearance of two n e u t r a l f r a c t i o n s at d i f f e r e n t p o s i t i o n s on the e l u t i n g g r a d i e n t r a i s e d the p o s s i b i l i t y of the presence of two n e u t r a l p r i n c i p l e s . The p r o p e r t i e s o f the two f r a c t i o n s were compared c a r e f u l l y , and the r e s u l t s w i l l be c o n s i d e r e d i n S e c t i o n C7, page ikl . A pure p r e p a r a t i o n o f o x y t o c i c p r i n c i p l e had been o b t a i n e d from the skate R a i a o c e l l a t a by the above p r o c e d u r e s ( P e r k s and Sawyer, 1 9 6 5 ) and i t was a n t i c i p a t e d t h a t the n e u t r a l p r i n c i p l e ( s ) from Squalus might a l s o be pure at t h i s s t a g e . T h e r e f o r e , a sample o f each of the two f r a c t i o n s was 132 Fi g u r e 9- Chromatogram of p a r t i a l l y p u r i f i e d n e u r o i n t e r -mediate lobe e x t r a c t of Squalus acanthias ( P r e p a r a t i o n 3; Sephadex G-15 p u r i f i e d ) o n a 35 cm x 0.9 cm column of CM Sephadex. The a c t i v i t y (22, 701 mU r a t uterus a c t i v i t y , without Mg + +) was a p p l i e d i n 4,364 ml of s o l u t i o n at pH 5.0 and s p e c i f i c c o n d u c t i v i t y 0 . l 6 mmho/cm, and was e l u t e d with WH^acetate b u f f e r of i n c r e a s i n g c o n c e n t r a t i o n , from 0.002M (pH 5.0): 0.05 M NH^acetate (pH 5.0) was int r o d u c e d i n t o the b u f f e r r e s e r v o i r at tube 2, and 0.20 M NH^acetate (pH 7-0) was intr o d u c e d at tube 153. S o l i d c i r c l e s = s p e c i f i c c o n d u c t i v i t y of the e l u a t e . Empty c i r c l e s = r a t uterus a c t i v i t y , without Mg + +: f r a c t i o n 1= 9 ,392 mU; f r a c t i o n 2 = 7,036 mU; f r a c t i o n 3 = 446 mU. Tube volumes = 3.4 ml. Table XIVJ The Chromatography of Squalus acanthias and Rai a r h i n a Neurohypophysial p r i n c i p l e s on CM Sephadex, F o l l o w i n g P u r i f i c a t i o n on Sephadex G-15 and DEAE Sephadex A c t i v i t y a p p l i e d to column E l u a t e P r e p a r a t i o n Load, mU(RU) a N e u t r a l p r i n c i p l e s Basic p r i n c i p l e T o t a l recov-ery of r a t uterus a c t i v i t y Peak 1 Peak 2 Peak 3 mU(RU) mU(RU) mU(RU) mU(ADH) b Squalus 2 25 ,016 >10 ,339 5 , 0 6 l - 1 , 2 6 l -Squalus 3 22 ,701 9,392 7,036 763 Squalus h 31 ,086 15 ,168 7,127 U53 - 13% Raia 1,827 356 652 - 209 -(RU) = r a t uterus a c t i v i t y , i n absence of magnesium. (ADH) = r a t a n t i d i u r e t i c a c t i v i t y . 135 hydrolyzed and subjected to amino a c i d a n a l y s i s so that i t s p u r i t y could he estimated. The r e s u l t s are given i n Table XV, column A. Each h y d r o l y s a t e contained more than the nine amino a c i d r e s i d u e s that could be expected f o r a p u r i f i e d neurohypo-p h y s i a l p r i n c i p l e . Therefore i t was necessary to subject each f r a c t i o n to a f u r t h e r p u r i f i c a t i o n procedure, which w i l l be d e s c r i b e d i n S e c t i o n C6 , page 137* c. The pharmacological a n a l y s i s of the b a s i c peak The t h i r d , b a s i c , f r a c t i o n e l u t e d from the CM Sephadex contained approximately 2% of the t o t a l r a t uterus a c t i v i t y i n the e l u a t e (see Table XIV). The small amount present pre-vented any attempt to o b t a i n a chemical a n a l y s i s of the amino a c i d composition of t h i s peak, and t h e r e f o r e s t u d i e s were con f i n e d to a pharmacological a n a l y s i s . A p r i n c i p l e which was p h a r m a c o l o g i c a l l y s i m i l a r to a r g i n i n e v a s o t o c i n had been demonstrated e a r l i e r by paper chromatography ( S e c t i o n C2c, page 125; a l s o see Table XVI). An attempt was made here to confirm t h i s i d e n t i f i c a t i o n . The t h i r d peak from the CM Sephadex was assayed against syn-t h e t i c o x y t o c i n (Syntocinon) f o r r a t uterus and f r o g bladder a c t i v i t i e s , and against v a s o p r e s s i n ( P i t r e s s i n ) f o r a n t i d i u r -e t i c a c t i v i t y . These a c t i v i t i e s are l i s t e d i n Table XVI, p a r t A. The r e s u l t i n g a c t i v i t y r a t i o s , R^^. = 2.0 and R ^ = 1 7 7 , were again comparable to those of a r g i n i n e v a s o t o c i n . For more c o n c l u s i v e evidence the ; Squalus p r i n c i p l e was Table X V Amino Acid Analyses of Squalus acanthias Neutral Fraction 1 and Neutral Fraction 2 at Two Stages of Purification Amino acid Molar ratio to glutamic acid  A B Purified on CM Sephadex Purified on both CM Sephadex and SE Sephadex Fraction l a Fraction 2 Fraction 1 Fraction 2 Neutral and acidic amino acids Cysteic acid 0.13 0.26 0.52 Ethanolamine +veD —ve -ve —ve Aspartic acid 1.93 < 1.97 3.30 2.33 Threonine 0.26 0.21 0.35 0.32 Serine 0.79 0.94 1.95 1.14 Glutamic acid 1.00 1.00 1.00 1.00 Proline 0.74 0.35 2.13 1.38 Half cystine —ve -ve —ve 0.83 Glycine 7.93 1.56 6.21 2.62 Alanine 0.17 -ve 0.89 0.75 Valine 0.62 0.50 0.73 0.95 Methionine 0.44 -ve 0.08 —ve Isoleucine 0.75 0.55 1.21 1.13 Leucine 0.90 0.56 1.07 0.80 Tyrosine 0.49 0.59 0.97 0.36 Phenylalanine 0.18 0.49 —ve -ve Basic amino acids Lysine 2.13 0.90 1.59 0.30 Histidine —ve 0.55 0.04 -ve Arginine 7.28 14.86 —ve -ve Ammonia +ve +ve +ve +ve Total amino acids Micromoles per unit oxytocic activity Neutral and acidic 0.0940 0.0822 0.0847 0.0699 Basic 0.0443 0.1515 0.0045 0.0015 Samples used for analyses (oxytocic activity, without Mg ): A. Purified on CM Sephadex: Fraction 1: from Preparation 3. Neutral and acidic amino acid values are the average values from two analyses - - of 836 mU and of 1255 mU. Basic amino acid values are from one analysis of 418 mU. Fraction 2: from Preparation 3. Neutral and acidic amino acid values were determined from 1327 mU; basic amino acid values, from 1327 mU. B. Purified on CM and SE Sephadex: Fraction 1: neutral fractions la and lb (fraction 1 from Preparations 2 and 3). Neutral and acidic amino acid values were determined by averaging the mean values from two analyses - - of 1235 mU and of 722 mU (neutral fraction la)- with the values from one analysis of 1180 mU (neutral fraction lb) Basic amino acid values are the average values from two analyses - of 542 mU (neutral fraction la) and of 1062 mU (neutral fraction lb) . . . . Fraction 2: neutral fraction 2a (fraction 2 from Preparations 2 and 3, combined). Neutral and acidic amino acid values were determined from 1441 mU; basic amino acid values, from 1441 mU. +ve: present; molar ratio not calculated. —ve: not detected during analysis. 137 then assayed d i r e c t l y a gainst s y n t h e t i c a r g i n i n e v a s o t o c i n f o r r a t uterus a c t i v i t y , with and without magnesium i o n s , f o r f r o g bladder a c t i v i t y , and f o r a n t i d i u r e t i c a c t i v i t y . Table XVI, part. B shows that the a c t i v i t y of the Squalus p r i n c i p l e r e l a t i v e to a r g i n i n e v a s o t o c i n was constant on a l l four assays, with the r e s u l t i n g a c t i v i t y r a t i o s - = 0.9^ ± 0.16, D = 0.96, and D A r i T T = 0.89 ± 0.21. This provided strong evidence f o r the s i m i l a r i t y of the Squalus p r i n c i p l e and a r g i n i n e v a s o t o c i n : since a r g i n i n e v a s o t o c i n and 8 - l y s i n e o x y t o c i n are alone i n t h e i r high f r o g bladder a c t i v i t y but are d i s t i n g u i s h e d from each other by the higher a n t i d i u r e t i c a c t i v i t y of a r g i n i n e v a s o t o c i n , i t i s almost c e r t a i n t h a t the value of D-p-g would have been l e s s than 1.0 i f the Squalus p r i n c i p l e were n e i t h e r a r g i n i n e v a s o t o c i n nor 8 - l y s i n e o x y t o c i n , and that the value of D would have been l e s s than 1.0 i f i t AD H were 8 - l y s i n e o x y t o c i n r a t h e r than a r g i n i n e v a s o t o c i n . N e i t h e r the value f o r D nor D g of the Squalus p r i n c i p l e d i f f e r e d from 1.0, and so i t s i d e n t i t y with a r g i n i n e v a s o t o c i n i s h i g h l y probable. 6. P u r i f i c a t i o n of the Two N e u t r a l F r a c t i o n s on  SE Sephadex The two n e u t r a l peaks from each CM.rSephadex column were l y o p h i l i z e d s e p a r a t e l y f o r the removal of ammonium b u f f e r s a l t s . The d r i e d r e s i d u e s of peaks 1. and 2 from P r e p a r a t i o n s 2 and 3 were s t o r e d at -l8°C f o r p e r i o d s of up to 5 months. • Between k0% and 80% of the o x y t o c i c a c t i v i t y was l o s t from these samples, and so i t was necessary to combine the Table XVI The A c t i v i t i e s and A c t i v i t y Ratios of the B a s i c Neurohypophysial P r i n c i p l e from Squalus ac a n t h i as Basic p r i n c i p l e A Assayed against normal standards' A c t i v i t i e s 1 3 (mU/ml) A c t i v i t y r a t i o s RUC ADH FB R ADH R FB CM Sephadex-purified ( P r e p a r a t i o n 3 , Peak 3 ) 3 0 . 4 59.2 5375 2.0 177 E l u t e d from paper , R J P 0 . 3 - 0 . 4 ( P r e p a r a t i o n 2) 4.5 12 2136 2.7 475 A r g i n i n e v a s o t o c i n Sawyer (1965) Munsick et a l . (i960) 1.6 2.0 910 271 B Assayed against s y n t h e t i c a r g i n i n e v a s o t o c i n A c t i v i t i e s 1 3 (ng/ml) A c t i v i t y r a t i o s RU RU+Mg ADH FB D Mg 'ADH D FB CM Sephadex-purified (Preparations 1, 2, 3 196 184 175 189 0.94 O..89 O.96 mixed; Peak 3) ± 2 1 ± 27 ± 3 8 ± 0.l6 ± 0.21 Table XVI continued Normal standards = s y n t h e t i c o x y t o c i n (Syntocinon, Sandoz; 10 IU/ml) f o r r a t uterus and f r o g bladder assays, or a r g i n i n e / l y s i n e v a s o p r e s s i n ( P i t r e s s i n , Parke Davis; 20 IU/ml) f o r the r a t a n t i d i u r e t i c assay. Assays c a l c u l a t e d by the 4-point method of Holton (1948); whenever three or more groups were obtained the f i d u c i a l l i m i t s are expressed, P = 0.05. °Abbreviations: RU, RU+Mg, ADH; as i n Table X. FB = a c t i v i t y on f r o g bladder assay. A c t i v i t y r a t i o s , R , R„_, D M , D.^^, and D^^, are the r a t i o s of the a c t i v i t y ADH r B Mg ADH r r> found on each assay (RU+Mg, ADH, FB) to the a c t i v i t y found on the r a t uterus assay (RU); where R i n d i c a t e s t h at the assays were performed against the normal, standards (footnote a ) , and D i n d i c a t e s that the assays were performed a g a i n s t a d i f f e r e n t peptide ( a r g i n i n e v a s o t o c i n ) . H vo Iho corresponding peaks from the two columns. Peak 1 from P r e p a r a t i o n 3 and part of peak 1 from P r e p a r a t i o n 2 were combined as " n e u t r a l f r a c t i o n l a " (3,055 mU r a t uterus a c t i v i t y , no M g + + ) . The remainder of peak 1 from P r e p a r a t i o n 2 (approximately 2 ,kl0 mU) became " n e u t r a l f r a c t i o n l b " . Peak 2 from P r e p a r a t i o n s 2 and 3 were combined as " n e u t r a l f r a c t i o n 2a" (3,513 mU). The two peaks from P r e p a r a t i o n k had not been s t o r e d a f t e r l y o p h i l i z -a t i o n , and only 22% and 5$ of the o x y t o c i c a c t i v i t y had been l o s t from peak 1 and peak 1, r e s p e c t i v e l y , by the fr e e z e d r y i n g process i t s e l f . Peak 1 became " n e u t r a l f r a c t i o n l c " ( l l , 8 l 4 mU) and peak 2 became " n e u t r a l f r a c t i o n 2b" (6,782 mU). The amino a c i d analyses of the CM Sephadex e l u a t e s (Table XV, column A) had shown that at t h i s stage of p u r i f i -c a t i o n n e i t h e r f r a c t i o n 1 nor f r a c t i o n 2 contained a pure neurohypophysial p e p t i d e . Wilson (1968) had found that e l u t i o n from two c a t i o n exchangers, at two d i f f e r e n t pH's , was necessary f o r the p u r i f i c a t i o n of i s o t o c i n from the salmon Onchorhynchus t schawyt scha. Therefore the Squalus p r i n c i p l e s were subjected to f u r t h e r p u r i f i c a t i o n on SE Sephadex, at a pH of 2.^5. The three samples of n e u t r a l f r a c t i o n 1 and the two samples of f r a c t i o n 2 were s e p a r a t e l y a d j u s t e d to pH 2.^5 and s p e c i f i c c o n d u c t i v i t y I.85 mmho/cm by dropwise a d d i t i o n of formic a c i d and d i l u t i o n with water to volumes ranging from 63 ml to 219 ml. Each was a p p l i e d to a separate 35 cm x 0.9 cm column of SE Sephadex which had been e q u i l i b r a t e d with O.lk M NH^ formate (pH 2.1+5). The a c t i v i t y was adsorbed onto the g e l and e l u t e d o f f on an NH.^formate g r a d i e n t between O.lk M (pH 2.45) and 1.5 M (pH 2.45), as shown i n Figure 1 0 . The recovery of o x y t o c i c a c t i v i t y from the g e l was almost complete, between 92% and 9 7 $ . A f t e r t h e i r e l u t i o n from SE Sephadex, samples of each of the two n e u t r a l f r a c t i o n s were l y o p h i l i z e d s e p a r a t e l y f o r the removal of ' NH^formate, h y d r o l y z e d , and analyzed f o r amino a c i d content. The analyses are l i s t e d i n Table XV, column B . They showed t h a t the main e f f e c t of the SE Sephadex was to s e p a r a t e . b a s i c amino a c i d r e s i d u e s away from the neurohypo-p h y s i a l p r i n c i p l e s . The data show that 9 0 - 9 9 $ of the b a s i c amino a c i d r e s i d u e s , s t i l l present i n each f r a c t i o n a f t e r e l u t i o n from CM Sephadex, were removed by the SE Sephadex. The SE exchanger a l s o removed 10%-15% of the n e u t r a l and a c i d i c amino a c i d s present a f t e r p u r i f i c a t i o n on CM Sephadex. Methionine and ethanolamine were removed from f r a c t i o n 1 and phen y l a l a n i n e from both f r a c t i o n s . The analyses of the two f r a c t i o n s were q u a l i t a t i v e l y s i m i l a r and both i n d i c a t e d more than the a n t i c i p a t e d nine r e s i d u e s of a neurohypophysial pep-t i d e . Thi.s w i l l be considered again i n Sect i o n s C7f and C 8 b , pages 151 and 1 5 8 . 7. Comparative Studies of the Two N e u t r a l F r a c t i o n s The repeated e l u t i o n of two n e u t r a l peptide f r a c t i o n s from the CM Sephadex r a i s e d the p o s s i b i l i t y that two d i s t i n c t n e u t r a l p r i n c i p l e s were e l a b o r a t e d by t h i s s p e c i e s . A 142 Figu r e 10a. Chromatogram of Squalus acanthias n e u t r a l f r a c t i o n l a ( f r a c t i o n 1 from P r e p a r a t i o n s 2 and 3, combined; CM Sephadex-purified) on a 35 cm x 0.9 cm column of SE Sephadex. The sample (3,055 mU r a t uterus a c t i v i t y , without Mg + +) was a p p l i e d i n 170 ml of s o l u t i o n at pH 2.45 and s p e c i f i c conduc-t i v i t y 1.85 mmho/cm, and was e l u t e d with NH^formate b u f f e r of i n c r e a s i n g concentra-t i o n , from 0.14 M (pH 2.45): 1.5 M NH^formate (pH 2.45) was i n t r o d u c e d i n t o the b u f f e r r e s e r v o i r at tube 1. S o l i d c i r c l e s = s p e c i f i c c o n d u c t i v i t y of the e l u a t e . Empty c i r c l e s = r a t uterus ac-t i v i t y , without Mg + +. Recovery of a c t i v i t y = 95%. Tube volumes = 3 . 4 ml. Fi g u r e 10b. Chromatogram of Squalus acanthias n e u t r a l f r a c t i o n 2a ( f r a c t i o n 2 from P r e p a r a t i o n s 2 and 3, combined; CM Sephadex-purified) on a 35 cm x 0.9 cm column of SE Sephadex. The sample (3,513 mU r a t uterus a c t i v i t y , without Mg + +, i n a volume of 110 ml) was a p p l i e d to the column and e l u t e d under the same c o n d i t i o n s as f r a c t i o n l a (see above). S o l i d c i r c l e s = s p e c i f i c c o n d u c t i v i t y of the e l u a t e . Empty c i r c l e s = r a t uterus a c t i v i t y , without Mg + +. Recovery of a c t i v i t y = 92%. Tube volumes = 3 . 4 ml. Spec i f ic c o n d u c t i v i t y , mmho/cm ihk d e m o n s t r a t i o n o f f u r t h e r d i f f e r e n c e s b e t w e e n t h e two f r a c t i o n s w o u l d v e r i f y p r e v i o u s r e p o r t s o f two n e u t r a l p r i n c i p l e s i n t h i s s p e c i e s ( H e l l e r and P i c k e r i n g , 1961). T h i s was i m p o r t a n t , s i n c e t h e o c c u r r e n c e o f more t h a n one n e u t r a l p e p t i d e w i t h i n any s p e c i e s i s r a r e . T h e r e f o r e a c a r e f u l c o m p a r i s o n o f t h e p r o p e r t i e s o f t h e two f r a c t i o n s was made. The r e s u l t s w i l l be d i s c u s s e d i n t u r n . a. The c o m p a r i s o n o f t h e p h a r m a c o l o g i c a l a c t i v i t i e s o f t h e n e u t r a l p e a k s , by a s s a y  a g a i n s t s y n t h e t i c o x y t o c i n ( S y n t o c i n o n ) E a c h o f t h e two f r a c t i o n s was a s s a y e d a g a i n s t s y n t h e t i c o x y t o c i n f o r r a t u t e r u s a c t i v i t y , b o t h w i t h and w i t h o u t mag-n e s i u m i o n s , f o r m i l k - e j e c t i o n a c t i v i t y , and f o r f r o g b l a d d e r a c t i v i t y . B o t h f r a c t i o n s were a s s a y e d t o g e t h e r on t h e same, p r e p a r a t i o n s . T a b l e X V I I shows t h a t t h e i r r e l a t i v e a c t i v i t i e s were s i m i l a r . T h e r e was no d i f f e r e n c e b e t w e e n t h e magnesium p o t e n t i a t i o n s ( R M g ) o f t h e two f r a c t i o n s , o r b e t w e e n t h e i r r a t i o s o f m i l k - e j e c t i o n t o r a t u t e r u s a c t i v i t y (R..^ ) . T h i s was s i g n i f i c a n t s i n c e t h e s e two r a t i o s have been u s e d t o d i s -t i n g u i s h b e t w e e n o t h e r e l a s m o b r a n c h p r i n c i p l e s ( s e e T a b l e I I I , page 3 3 ) . The a p p a r e n t d i f f e r e n c e b e t w e e n t h e two r a t i o s o f f r o g b l a d d e r a c t i v i t y t o r a t u t e r u s a c t i v i t y (R,-,,,) c a n n o t be r e g a r d e d as s i g n i f i c a n t , s i n c e e a c h e s t i m a t e o f f r o g b l a d d e r a c t i v i t y was b a s e d on o n l y one r e s p o n s e t o t h e unknown. b. The c o m p a r i s o n o f t h e p h a r m a c o l o g i c a l a c t i v i t i e s o f t h e n e u t r a l p e a k s , by a s s a y  o f one f r a c t i o n a g a i n s t t h e o t h e r The two f r a c t i o n s were a s s a y e d d i r e c t l y a g a i n s t e a c h Table XVII The A c t i v i t i e s and A c t i v i t y Ratios of the Two Squalus acanthias N e u t r a l F r a c t i o n s 8 , from F r a c t ion A c t i v i t i e s (mU/ml) A c t i v i t i e s r a t i o s RU C RU+Mg ME FB R Mg RME R FB 1 58.7 ± 8.0 120 . 3 ± 34.0 107.1 ± 58.1+ 1+05 2.1 ± 0.6 1.8 ± 1.0 6.9 2 I+9.9 ± 6.2 115.8 ± 3 1 . 4 86.1 ± 67.1 860 2 . 3 ± 0.1 l . T 1 1 . 3 17.2 CM Sephadex eluates ( P r e p a r a t i o n 3 ) . ^Assays c a l c u l a t e d by the 4-point method of Holton (1948); whenever three or more groups were obtained, the f i d u c i a l l i m i t s are expressed, P = 0.05. A b b r e v i a t i o n s as i n Table X and XVI. 146 o t h e r , again f o r r a t uterus a c t i v i t y , "both with and without magnesium i o n s , f o r m i l k - e j e c t i o n a c t i v i t y , and a l s o f o r a n t i d i u r e t i c a c t i v i t y . Table XVIII- shows that the a c t i v i t y of f r a c t i o n 2 r e l a t i v e to the a c t i v i t y of f r a c t i o n 1 was constant on a l l four assays, as would be expected i f the two p r i n c i p l e s were i d e n t i c a l . Thus no d i f f e r e n c e was demonstrated between the two f r a c t i o n s by pharmacological comparison, e i t h e r by assay side by side a gainst s y n t h e t i c o x y t o c i n , or by the d i r e c t assay of one f r a c t i o n a g ainst the o t h e r , a method which e l i m i -nates e r r o r s due to v a r i a t i o n s i n the s e n s i t i v i t y of assay p r e p a r a t i o n s to standards or unknowns. c . Paper chromatography of the two f r a c t i o n s The two f r a c t i o n s were a p p l i e d to adjacent o r i g i n s on Whatman 3MM paper and the chromatogram was developed i n n - b u t a n o l : a c e t i c acidrwater = 4:1:5. The e l u a t e s were assayed on the r a t uterus i n the presence of magnesium, and the r e s u l t s are shown i n Figure 5» e and f , page HV?,".. Both f r a c t i o n s e x h i b i t e d the same behavior and.were l o c a t e d i n R„ 0.4-0.7. Since n e i t h e r occupied the p o s i t i o n of the p r i n c i p l e E^ , which i s slow-running i n t h i s solvent system, i t i s apparent that the two Squalus f r a c t i o n s found here d i d not correspond to the p r i n c i p l e s E^ and Eg which had been r e p o r t e d e a r l i e r i n t h i s species ( H e l l e r and P i c k e r i n g , 1 9 6 l ) . No d i f f e r e n c e between the two Squalus f r a c t i o n s was demonstrated by t h i s procedure. Table X V I I I 9J The A c t i v i t i e s and A c t i v i t y Ratios of Squalus acanthias N e u t r a l F r a c t i o n 2 , Assayed against N e u t r a l F r a c t i o n l a Volume A c i t v i t i e s - - e q u i v a l e n t A c t i v i t y r a t i o s of volume (ml) of f r a c t i o n 1 f r a c t i o n 2 — RU C RU+Mg ME ADH, D„ D„_, D A T,„ B Mg ME ADH 1 ml 0.70 0.65 0.75 0.68 0.9b 1.08 0..97 ± 0.10 ±0.09 ±0.18 ±0.34 ±0.18 ± 0.26 ±0.49 aSE Sephadex eluates ( P r e p a r a t i o n 4 ) . Assays c a l c u l a t e d by the 4-point method of Holton (1948); whenever three or more groups were obtained the f i d u c i a l l i m i t s are expressed, P = 0.05. c A b b r e v i a t i o n s as i n Table . X; except the a c t i v i t y r a t i o s , D^ , D^, and D^pg, where D i n d i c a t e s that the assays were not performed against the^normal standards. IhQ d. I s o e l e c t r i c f o c u s i n g of the two f r a c t i o n s A mixture of the two f r a c t i o n s was subjected to i s o -e l e c t r i c f o c u s i n g , a s e n s i t i v e technique which can separate compounds d i f f e r i n g i n i s o l e c t r i c p o i n t by as l i t t l e as 0 . 0 2 pH u n i t s (Vesterberg and Svensson, 1 9 6 6 ) . This method had not p r e v i o u s l y been a p p l i e d i n the study of neurohypophysial p r i n c i p l e s . I t i s an important technique, i n view of the s i m i l a r i t y of these peptides and of the d i f f i c u l t y i n d i s -t i n g u i s h i n g them by other means. A sample of kOO mU o x y t o c i c a c t i v i t y ( 2 0 0 mU from each f r a c t i o n ) of SE Sephadex-purified m a t e r i a l was subjected to 300 v o l t s f o r Uo hours i n a pH range 3 - 1 0 ; and a second sample of 3 9 1 mU ( 2 6 l mU from f r a c t i o n 1 , 130 mU from f r a c t i o n 2 ) of CM S e p h a d e x - p u r i f i e d m a t e r i a l was subjected to 8 0 0 v o l t s f o r 2 5 hours i n a narrower pH range of 7 - 9 - In each case h0% of the a c t i v i t y was recovered from the column. Once again, there was no d i f f e r e n c e i n the behavior of the two f r a c t i o n s . They were not r e s o l v e d i n e i t h e r run; they migrated i n the pH g r a d i e n t as one peak, and s t a b i l i z e d at pH 7 . 6 2 ( F i g . 1 1 ) . e. The comparison of the behavior of the two  f r a c t i o n s on SE Sephadex Since the two n e u t r a l f r a c t i o n s had been e l u t e d from the CM Sephadex at d i f f e r e n t b u f f e r c o n c e n t r a t i o n s , t h e i r behavior on SE Sephadex, a l s o a c a t i o n exchanger, was of i n t e r e s t . However they appeared s i m i l a r i n t h e i r behavior ihg F i g u r e 11. I s o e l e c t r i c f o c u s i n g of a mixture of Squalus acanthi as n e u t r a l f r a c t i o n 1 and n e u t r a l f r a c t i o n 2 ( P r e p a r a t i o n 3, Sephadex G-15- and CM Sephadex-purified) i n a pH range of 7-9 at 800 v o l t s f o r 25 hours. Load = 391 mU r a t uterus a c t i v i t y , without Mg + + (26l mU of f r a c t i o n 1 and 130 mU of f r a c t i o n 2). S o l i d c i r c l e s = pH of e l u a t e . Empty c i r c l e s = o x y t o c i c a c t i v i t y ( r a t uterus a c t i v i t y , without M g + + ) . Recovery of a c t i v i t y = k0%. Tube volumes = 1.1 ml. 15 0 Squalus acanthias, isoelectric focusing Tube number 151 on t h i s exchanger (see S e c t i o n C6 above , page 137). Both were e l u t e d i n the same p o s i t i o n on the NH^formate g r a d i e n t , as i s i l l u s t r a t e d i n Figu r e 10. The three samples of f r a c t i o n 1 appeared w i t h i n the s p e c i f i c c o n d u c t i v i t i e s of 5.8 and 11.6 mmho/cm and the two samples of f r a c t i o n 2 were e l u t e d between 6.9 and 11.8 mmho/cm. f. The comparison of the amino a c i d composition  of the two f r a c t i o n s A f t e r p u r i f i c a t i o n on SE Sephadex a sample of each f r a c t i o n was hydrolyzed and analyzed f o r amino a c i d composi-t i o n ( S e c t i o n C6 above). The r e s u l t s were l i s t e d i n Table XV, column B (page 136). The analyses of the two f r a c t i o n s were q u a l i t a t i v e l y s i m i l a r . F r a c t i o n 1 contained r e l a t i v e l y more g l y c i n e than f r a c t i o n 2 , and there were d i f f e r e n c e s between t h e i r t y r o s i n e and c y s t e i c a c i d contents. However the ex t r a g l y c i n e i n f r a c t i o n 1 was l a t e r removed by rechromatography on CM Sephadex (see S e c t i o n g below), and so i t d i d not form a part of the a c t i v e p e p t i d e . The recovery of t y r o s i n e and c y s t e i c a c i d from the h y d r o l y s i s procedure i s known to vary (Chauvet e_t al_. , 1963) and so q u a n t i t a t i v e d i f f e r e n c e s i n these amino a c i d r e s i d u e s may not i n d i c a t e any r e a l d i f f e r e n c e between the peptides i n the two f r a c t i o n s . g. Rechromatography of f r a c t i o n 1 on CM Sephadex The two ox y t o c i c f r a c t i o n s had been d i s t i n g u i s h e d by the b u f f e r c o n c e n t r a t i o n s at which they were e l u t e d from CM Sephadex. However, a f u r t h e r d i f f e r e n c e between them could 152 not be demonstrated, even by such d i v e r s e means as pharmaco-l o g i c a l comparisons, paper chromatography, i on exchange chromatography, i s o e l e c t r i c f o c u s i n g , or comparisons of t h e i r amino a c i d compositions. ( S e c t i o n a - f , above). Therefore an attempt was made to demonstrate the i d e n t i t y of the two p r i n c i p l e s , by rechromatography of f r a c -t i o n 1 on CM Sephadex. A f t e r i t s e l u t i o n from SE Sephadex a + + sample of f r a c t i o n 1 (7,900 mU r a t uterus a c t i v i t y , no Mg pre s e n t , from P r e p a r a t i o n k) was l y o p h i l i z e d f o r the removal of NH^formate. The res i d u e ( c o n t a i n i n g 6,400 mU r a t uterus ++ a c t i v i t y i n the absence of Mg , a recovery of Ql% , and a l s o •. a l i t t l e remaining e l e c t r o l y t e ) was taken up i n 100 ml water. The s p e c i f i c c o n d u c t i v i t y of the r e s u l t i n g s o l u t i o n was 0.l6 mmho/cm and the pH was 5.0. I t was a p p l i e d , without f u r t h e r adjustment, to a 35 cm x 0.9 cm column of CM Sephadex, Lot 8852, which had been e q u i l i b r a t e d with 0.002 M HN^acetate (pH 5-0), the same c o n d i t i o n s as p r e v a i l e d during the f i r s t chromatography of P r e p a r a t i o n 4 on CM Sephadex. However P r e p a r a t i o n h had been a p p l i e d to the f i r s t CM Sephadex column i n a l a r g e r volume, 2,485 ml. Peak 1 of P r e p a r a t i o n k had come o f f the f i r s t CM Sephadex column between the s p e c i f i c c o n d u c t i v i t i e s of 0.l6 mmho/cm and 6.2 mmho/cm, while peak 2 had appeared only a f t e r the s p e c i f i c c o n d u c t i v i t y had reached 6.2 mmho/cm ( F i g . 12a). This time, when f r a c t i o n 1 was reloaded onto the r e s i n i n the much smal l e r volume of 100 ml, i t appeared i n the p o s i t i o n of 15 3 Fig u r e 12a. Chromatogram of p a r t i a l l y p u r i f i e d neuro-intermediate lobe e x t r a c t of Squalus  acanthias ( P r e p a r a t i o n k; Sephadex G-15-p u r i f i e d ) on a 35 cm x 0.9 cm column of CM Sephadex. The a c t i v i t y (31, 086 mU r a t uterus a c t i v i t y , without Mg + +) was a p p l i e d i n 2,485 ml of s o l u t i o n at pH 5.0 and s p e c i f i c c o n d u c t i v i t y 0.l6 mmho/cm, and was e l u t e d with NH^acetate b u f f e r of i n c -r e a s i n g c o n c e n t r a t i o n , from 0.002 M (pH 5.0): 0.10 M NH^acetate (pH 5.0) was int r o d u c e d i n t o the b u f f e r r e s e r v o i r at tube 3, and 0.20 M NH^acetate (pH 7.0) was int r o d u c e d at tube 97. Empty c i r c l e s = s p e c i f i c c o n d u c t i v i t y of the e l u a t e . Hatched columns = r a t uterus a c t i v i t y , without Mg + +: f r a c t i o n 1 = 15,l68 mU; f r a c t i o n 2 = 7,127 mU ; f r a c t i o n 3 = ^53 mU. Tube volumes = U.8 ml. Figure 12b. Chromatogram of Squalus ac a n t h i as f r a c t i o n 1 ( P r e p a r a t i o n h; p u r i f i e d on Sephadex G-15 and on CM Sephadex - see F i g . 12a, above -and a l s o on SE Sephadex) on a second 35 cmx 0.9 cm column of CM Sephadex. The a c t i v i t y {6 , k 0 0 mU r a t uterus a c t i v i t y , without Mg + +) was a p p l i e d i n 100 ml of s o l u t i o n at pH 5.0 and s p e c i f i c c o n d u c t i v i t y 0.l6 mmho/cm, and was e l u t e d with NH^acetate b u f f e r of i n c r e a s i n g c o n c e n t r a t i o n , from 0.002 M (pH 5-0): 0.10 M NH^acetate (pH 5.0) was intr o d u c e d i n t o the b u f f e r r e s e r v o i r at tube k. Empty c i r c l e s = s p e c i f i c c o n d u c t i v i t y of the e l u a t e . Hatched column = o x y t o c i c ac-t i v i t y ( r a t uterus a c t i v i t y , without M g + + ) ; 6,138 mU. Tube volumes = h.8 ml. 154 Squalus acanthiaSj chromatography on C M Sephadex o -E 1 8 J y -I C u o V Q 8. CO E 2 £ 1 2 E $ • > Q +-> 3 -C 4 0 -u u 1 °1 a to °t 0 - 1 0 M b u f f e r p H 5 . 0 Tube number f i o o 0 - 2 0 M b u f f e r p H 7 - 0 E 6 Q Squalus acanthias, Fraction 1, re -chromatography on C M Sephadex O x y t o c i c a c t i v i t y ) E l u a t e conductivity 0 - 1 0 M b u f f e r p H 5 - 0 6 0 Tube number 1 5 5 f r a c t i o n 2 , at a s p e c i f i c c o n d u c t i v i t y of 6 . 2 mmho/cm ( F i g . 1 2 b ) . On the b a s i s of t h i s experiment and the preceeding comparisons of the two f r a c t i o n s , i t was concluded that the neurohypophysial peptide contents of the two were i d e n t i c a l . The appearance of the n e u t r a l p r i n c i p l e at two d i f f e r e n t p o s i t i o n s on the e l u t i o n g r a d i e n t might be a t t r i b u t e d here to the l a r g e volumes i n which the samples were a p p l i e d to the exchanger ( 2 , 0 0 7 ml, 4 , 3 6 4 ml, 2 , 4 8 5 ml f o r P r e p a r a t i o n s 2 , 3 , and 4 , r e s p e c t i v e l y ) . It i s worth noting that the n e u t r a l pep-t i d e of the Rai a r h i n a e x t r a c t , t e n t a t i v e l y i d e n t i f i e d as g l u m i t o c i n alone ( S e c t i o n I B 3 and C 4 , pages 8 2 a n d - 9 8 ) was als o e l u t e d from CM Sephadex i n two f r a c t i o n s when the exchanger was loaded at a high volume, but that i t emerged as a s i n g l e peak when the two f r a c t i o n s were recombined and a p p l i e d to a second column i n a small volume. 8. The Nature of the S i n g l e N e u t r a l F r a c t i o n The preceeding experiments suggested that the Squalus e x t r a c t contained only one n e u t r a l f r a c t i o n . An attempt was made to determine i t s nature by pharmacological study and by the a n a l y s i s of i t s amino a c i d content. a. Pharmacological s t u d i e s of the n e u t r a l f r a c t i o n The a c t i v i t y r a t i o s f o r crude neurohypophysial e x t r a c t s of Squalus acanthias (Table X, page 1 0 6 ) had i n d i c a t e d that the o x y t o c i n - l i k e p r i n c i p l e was of the EOP I type. This was confirmed by the values f o r R of 1 . 8 ± 1 . 0 and 1 . 7 ± 1 - 3 156 found f o r " n e u t r a l f r a c t i o n l " and " n e u t r a l f r a c t i o n 2" a f t e r t h e i r s e p a r a t i o n on CM Sephadex (Table XVII, page 145). How-ever since g l u m i t o c i n had been i d e n t i f i e d i n four species of skate (Acher et_ a l . , 1965 ; 1967 ; Chauvet e_t a l . , I965) and had been t e n t a t i v e l y i d e n t i f i e d here i n the longnose skate, Rai a  r h i n a , ( S e c t i o n I B3 and I C4, pages 82, 98 ) i t was of i n t e -r e s t to know whether i t might a l s o be the p r i n c i p l e produced by the shark, Squalus a c a n t h i a s . This was not u n l i k e l y , since neurohypophysial e x t r a c t s of three of the skates found to pro-duce g l u m i t o c i n - Raia c l a v a t a , Raia b a t i s , and Raia r h i n a -had appeared to c o n t a i n p r i n c i p l e s of the EOP I type (Perks and Dodd, 1963a; data presented here i n S e c t i o n I BI, page 70). Therefore a p u r i f i e d p r e p a r a t i o n of the Squalus n e u t r a l p r i n c i p l e was assayed against s y n t h e t i c g l u m i t o c i n f o r r a t uterus a c t i v i t y , both i n the presence and absence of magnesium i o n s , and f o r m i l k - e j e c t i o n a c t i v i t y . The r e s u l t s are l i s t e d i n Table XIX. The potency of the Squalus p r i n c i p l e r e l a t i v e to g l u m i t o c i n was d i f f e r e n t on each of the three assays. The r a t i o of i t s a c t i v i t y on the r a t uterus i n the presence of Mg + + to i t s a c t i v i t y i n the absence of Mg + +, D w = 0.49 ± 0.06, Mg demonstrated the la c k of i d e n t i t y between the two p e p t i d e s . This was v e r i f i e d by the r a t i o of m i l k - e j e c t i o n a c t i v i t y to r a t uterus a c t i v i t y , D = 0.20. These r e s u l t s confirmed the f i n d -ings of Sawyer et_ al_. (1969 ) 5 who found that the p r i n c i p l e produced by Squalus a c a n t h i a s , A t l a n t i c v a r i e t y , was d i f f e r e n t from g l u m i t o c i n . 157 Table XIX The A c t i v i t i e s and A c t i v i t y Ratios of the Oxytocin-1ike F r a c t i o n 3 , from Squalus a c a n t h i as , Assayed against S y n t h e t i c G l u m i t o c i n A c t i v i t i e s (ng/ml) A c t i v i t y r a t i o s RU C RU + Mg ME DMg DME 23.h ± 1.9 11.5 ± 1 . 3 4.7 0.49 ± 0.06 0.20 aSE Sephadex e l u a t e of n e u t r a l f r a c t i o n l a (= Prepara-t i o n s 2 and 3, mixed; Peak l ) . Assays c a l c u l a t e d by the 4-point method of Holton (1948); whenever three or more groups were obta i n e d , the f i d u c i a l l i m i t s are expressed, P = 0.05. c A b b r e v i a t ions as i n Table X.; except the a c t i v i t y r a t i o s , D M and Djyjg , where D i n d i c a t e s that the assays were performed against a p r i n c i p l e ( g l u m i t o c i n ) other than the normal standard. 1 5 8 This s p e c i e s , then, appeared to produce a p r i n c i p l e other than the g l u m i t o c i n of the skates. I t might he that i t produces a mixture of g l u m i t o c i n and another p e p t i d e , so tha t the mixture appears p h a r m a c o l o g i c a l l y d i f f e r e n t from g l u m i t o c i n . However, chromatographic evidence (Sawyer, p e r s o n a l communication) and t h e o r e t i c a l c o n s i d e r a t i o n s (presented i n the D i s c u s s i o n , Chapter h) suggest that glumi-t o c i n would not he present i n any p o s s i b l e Squalus peptide mixture. A t h i r d p o s s i b i l i t y i s that the Squalus o x y t o c i n -l i k e f r a c t i o n c o n s i s t s of a mixture of p r i n c i p l e s , none of which i s g l u m i t o c i n . b. Amino a c i d analyses of the n e u t r a l f r a c t i o n A f u r t h e r attempt was made to determine the nature of the Squalus n e u t r a l p r i n c i p l e by an a n a l y s i s of i t s amino a c i d composition. S e v e r a l analyses were made; these w i l l be o u t l i n e d i n t u r n : i • The a n a l y s i s of " f r a c t i o n 2 " a f t e r  p u r i f i c a t i o n on SE Sephadex The amino a c i d analyses of the SE Sephadex-purified f r a c t i o n 1 and f r a c t i o n 2 (from combined P r e p a r a t i o n s 2 and 3 ; see Table XV, column B, page 1 3 6 ) suggested t h a t " f r a c t i o n 2 " was the purer p r e p a r a t i o n of the Squalus o x y t o c i n - l i k e p r i n c i p l e . The SE Sephadex had not removed the g l y c i n e which had been e l u t e d from the CM Sephadex i n t o " f r a c t i o n l " ; and a f t e r e l u t i o n from SE Sephadex, " f r a c t i o n l " contained 0 . 0 8 5 u mole of neu-t r a l and a c i d i c amino a c i d r e s i d u e s per u n i t of o x y t o c i c 159 a c t i v i t y , while " f r a c t i o n 2" contained only 0.070 u moles. Nevertheless " f r a c t i o n 2" contained more than the nine amino a c i d r e s i d u e s t y p i c a l of a s i n g l e neurohypophysial p r i n c i p l e . i i . The a n a l y s i s of the combined f r a c t i o n s An attempt was made to v e r i f y the above r e s u l t by an a n a l y s i s of m a t e r i a l which had been p u r i f i e d independently from the previous sample. F r a c t i o n 1 and f r a c t i o n 2 from P r e p a r a t i o n k (6.2 IU and 4.7 IU r a t uterus a c t i v i t y i n the absence of Mg + +, r e s p e c t i v e l y ) were combined a f t e r f r a c t i o n 1 had been rechromatogrammed on CM Sephadex (see S e c t i o n 7g above, page 151)• A sample of the combined m a t e r i a l was hydr o l y z e d and analyzed. The r e s u l t s are l i s t e d i n Table XX, column 2. The e x t r a g l y c i n e of the " f r a c t i o n 1" p o r t i o n (see Table XV, Column B) appeared to have been removed during i t s rechroma-tography on CM Sephadex. Table XX shows that t h i s a n a l y s i s of m a t e r i a l from P r e p a r a t i o n h was s i m i l a r to the previous a n a l y s i s of m a t e r i a l from combined P r e p a r a t i o n s 2 and 3 (Table XV, column B; repeated i n Table XX, column l ) . It was p o s s i b l e that some f r e e amino acids had not been separated from the neurohypophysial peptide by any.of the p u r i f i c a t i o n procedures which had been used. Therefore an unhydrolyzed sample of the combined f r a c t i o n s 1 and 2 from P r e p a r a t i o n h (6k2 mU r a t uterus a c t i v i t y , no Mg + + present) was analyzed f o r f r e e amino a c i d s . Only two could be detected,-s e r i n e and g l y c i n e (Table XXI), and the a n a l y s i s of the hydro-l y z e d sample of P r e p a r a t i o n h (Table XX, column 2) has been Table X X Amino Acid Analyses of the Purified Neutral Fraction from Squalus acanthias Amino acid Molar ratio to glutamic acid Fraction 2 Fractions 1 and 2, combined Average (CM and SE (CM, SE, and CM Sephadex-purified) values Sephadex-purified)3 (plus paper chromatography) 1 2 3 4 Neutral and acidic amino acids Cysteic acid 0.97b 0.84 1.56 1.12 Aspartic acid 2.33 1.94 2.21 2.16 Threonine 0.32 -ve c -ve 0.11 Serine 1.14 0.87d 1.00 1.00 Glutamic acid 1.00 1.00 1.00 1.00 Proline 1.38 1.63 2.87 1.96 Half cystine 0.83 -ve -ve _ Glycine 2.62 2.09d 2.28 2.33 Alanine 0.75 0.64 0.62 0.67 Valine 0.95 0.88 1.05 0.96 Methionine -ve -ve —ve —ve Isoleucine 1.13 1.01 1.08 1.07 Leucine 0.80 0.89 1.13 0.94 Tyrosine 0.36 0.29 -ve 0.22 Phenylalanine —ve 0.17 -ve 0.06 Basic amino acids Lysine 0.30 _ -ve Histidine —ve — -ve _ Arginine —ve — -ve _ Ammonia +ve - +ve -Total amino acids Micromoles per unit oxytocic activity Neutral and acidic 0.0699 0.0523 0.0685 Basic 0.0015 - -ve aSamples used for analyses: Column 1: see Table XV, footnote a: Fraction 2, purified on both CM and SE Sephadex. Column 2: combined neutral fractions lc and 2b (fractions 1 and 2 from Preparation 4); both fractions purified on CM and SE Sephadex, and fraction lc then rechromatogrammed on CM Sephadex. Neutral and acidic amino acid values were determined from 1280 mU oxytocic activity. Basic amino acid values were not determined. Column 3: combined neutral fractions lc and 2b (as in column 2), further purified by paper chromatography. All amino acids were determined from one sample, estimated to contain 603 mU oxytocic activity. ^Corrected to include the half cystine. c+ve: present; molar ratio not calculated, -ve: not detected during analysis. ^Corrected for the presence of serine and glycine in the unhydrolyzed sample (see Table XXI). Table XXI Amino A c i d A n a l y s i s of the Unhydrolyzed, P u r i f i e d N e u t r a l F r a c t i o n 8 , from Squalus acanthias Amino a c i d Micromoles i n Molar r a t i o to the unhydrolyzed glutamic a c i d i n sample (61+2 mU) the hydrolyzed sample (Table XX, column 2 ) b c C y s t e i c a c i d -ve A s p a r t i c a c i d -ve Threonine -ve Serine 0.0011 0.1+0 Glutamic a c i d -ve P r o l i n e -ve G l y c i n e 0.0011 0. 39 Alanine -ve V a l i n e -ve Methionine -ve I s o l e u c i n e -ve Leucine -ve T y r o s i n e -ve P h e n y l a l a n i n e - ve Combined n e u t r a l f r a c t i o n s 1 and 2 from P r e p a r a t i o n 1+; as i n Sample 2, Table XXV-' N e u t r a l and a c i d i c amino acids = a n a l y s i s of 61+2 mU of unhydrolyzed sample. Basic amino a c i d s not determined. Co r r e c t e d f o r the r a t i o of the unhydrolyzed sample to the hyd r o l y z e d sample (61+2 mU: 1280 mU). c -ve : not detected during a n a l y s i s . 162 c o r r e c t e d f o r these two r e s i d u e s . I t was a l s o p o s s i b l e that some contamination might have been i n t r o d u c e d i n the HC1 which was used f o r the h y d r o l y s e s . Therefore a sample of the HC1 was subjected to a blank h y d r o l y s i s procedure and then to amino a c i d a n a l y s i s . It was found to c o n t a i n s e v e r a l a c i d s , but they were i n small p r o p o r t i o n to the amounts of peptide t h a t were hy d r o l y z e d . Table XXII shows the low molar r a t i o of these contaminants to the glutamic a c i d found i n h y d r o l y -sates 1 and 2 of Table XX (column 1 and 2). Hydrolysate 3 (Table XX, column 3) was prepared with a d i f f e r e n t l o t of HC1 . i i i . The a n a l y s i s of the combined f r a c t i o n s  a f t e r paper chromatography It was p o s s i b l e that the procedures which were f o l -lowed had not succeeded i n s e p a r a t i n g a contaminating peptide away from the o x y t o c i c p r i n c i p l e . Therefore the remaining unhydrolyzed p o r t i o n of the combined f r a c t i o n s 1 and 2 from P r e p a r a t i o n k (3.6k IU r a t uterus a c t i v i t y i n the absence of Mg + +) was s u b j e c t e d to a d d i t i o n a l p u r i f i c a t i o n by a f u r t h e r method, based on d i f f e r e n t p r i n c i p l e s - by p a r t i t i o n chroma-tography, on paper, using n - b u t a n o l : a c e t i c acidrwater = 4:1:5. Oxytocic a c t i v i t y was l o c a t e d at R 0.25-0.60 and was e l u t e d r i n t o 0.25% a c e t i c a c i d . The e l u a t e was c e n t r i f u g e d f o r removal of c e l l u l o s e , and l y o p h i l i z e d . The r e s i d u e was hydro-l y z e d and analyzed f o r amino a c i d content. The r e s u l t s of the a n a l y s i s are l i s t e d i n Table XX, column 3. A comparison of the two analyses of the sample, before and a f t e r i t s paper chroma-163 Table XXII Amino A c i d A n a l y s i s of the H y d r o c h l o r i c A c i d Used f o r H y d r o l y s e s a Amino a c i d Micromoles Molar r a t i o to the g l u t a -i n k ml HC1 mic a c i d i n Hydrolysate. 1 Hydrolysate 2 (Table XX,- (Table. .XX, column 1) column 2) C y s t e i c a c i d c -ve A s p a r t i c a c i d 0.00063 0.08 0. 02 Threonine -ve -Serine 0.00015 0. 02 0.01 Glutamic a c i d 0.00013 0. 02 0. 01 P r o l i n e -ve - -G l y c i n e 0.00109 0. 14 0.04 Ala n i n e 0.00015 0. 02 0. 01 V a l i n e -ve - • .-Methionine -ve - -I s o l e u c i n e - ve - -Leucine -ve - -Tyro s ine -ve - -P h e n y l a l a n i n e -ve — — Lys ine ' O.OOO.7O 0.09 0.03 H i s t i d i n e - ve - -A r g i n i n e -ve — — a Used f o r a l l h y d r o l y s e s , except the h y d r o l y s i s of the sample p u r i f i e d by paper chromatography (Table XX, column 3)• Corrected f o r the p r o p o r t i o n of the t o t a l h y d r o l y s a t e which was used i n a n a l y s i s . °-ve: not detected during a n a l y s i s . 16k tography (Table XX, columns 2 and 3) shows that no f u r t h e r p u r i f i c a t i o n was achieved by t h i s step. i v . An i n t e r p r e t a t i o n of the analyses of the n e u t r a l f r a c t i o n I f an allowance i s made f o r the breakdown of c y s t i n e and t y r o s i n e during h y d r o l y s i s , these three s i m i l a r analyses of the p u r i f i e d m a t e r i a l (Table XX) suggest the presence of the amino a c i d r e s i d u e s of a mixture of two neurohypophysial p e p t i d e s , present i n equimolar amounts and having an average potency of 109 IU/mg. D e s t r u c t i o n of c y s t i n e and t y r o s i n e during h y d r o l y s i s i s known to occur (Chauvet et_ al_. , 1963) and i t i s p a r t i c u l a r l y marked when minimal amounts of peptide are present i n the h y d r o l y s i s mixture, as was the case here. A l l the n a t u r a l l y o c c u r r i n g neurohypophysial peptides c o n t a i n s i n g l e r e s i d u e s of asparagine , p r o l i n e , and g l y c i n e , i n ad-d i t i o n to c y s t i n e and t y r o s i n e (see Table I, page 6) and these three r e s i d u e s (asparagine h y d r o l y z e d to a s p a r t i c a c i d ) were found i n double p r o p o r t i o n i n the Squalus h y d r o l y s a t e . The s i x r e s i d u e s which were present i n s i n g l e p r o p o r t i o n i n the h y d r o l y s a t e - s e r i n e , glutamic a c i d , a l a n i n e , v a l i n e , i s o l e u -c i n e , and l e u c i n e - could occupy the remaining three p o s i t i o n s i n each of two peptide molecules; i t i s the amino acids of these p o s i t i o n s , p o s i t i o n 3, h, and 8, which have been found to vary i n nature. 165 D. Summary of the Studies on the Neurohypophysial P r i n c i p l e s of Squalus acanthias 1. E a r l y pharmacological, and chromatographic s t u d i e s suggested that Sgualus acanthias produces only an o x y t o c i n -l i k e p r i n c i p l e (Perks et_ al_. , i960 ; Sawyer e_t_ a_l. , 196I; Perks and Dodd, 1963a; Perks, 1966). However i t was found here, i n agreement with Sawyer (1965; and l a t e r , 1967) that Sq.ualus e l a b o r a t e d a .small amount of b a s i c p e p t i d e , which accounted f o r approximately 2% of the t o t a l r a t uterus a c t i v -i t y of the e x t r a c t , i n a d d i t i o n to the o x y t o c i c f r a c t i o n . The b a s i c p r i n c i p l e was i d e n t i f i e d as a r g i n i n e v a s o t o c i n on the b a s i s o f : a. I t s m o b i l i t y on paper chromatography ( F i g . 8, page 128). b. I t s pharmacological agreement with a r g i n i n e v a s o t o c i n , both ( i ) when assayed by the c l a s s i c a l methods against s y n t h e t i c o x y t o c i n (Syntocinon) and v a s o p r e s s i n ( P e t r e s s i n ) , (Table XVIA, page 138): and, more c o n c l u s i v e l y , ( i i ) when assayed d i r e c t l y a gainst s y n t h e t i c a r g i n i n e vaso-t o c i n (Table XVIB). 2. a. The n e u t r a l f r a c t i o n was e l u t e d from the CM Sephadex i n two peaks ( F i g . 9, page 133). This r a i s e d the p o s s i b i l i t y of the presence of two n e u t r a l p r i n c i p l e s w i t h i n a s i n g l e species - a r a r e occurrence. However, a c a r e f u l comparison of the two peaks by pharmacological s t u d i e s (Tables XVII, XVIII, pages 145, 1^7 )» "by paper chromatography 166 ( F i g . 5 e , f j page 115), by ion exchange chromatography on SE Sephadex ( F i g . 10, page 143), by i s o e l e c t r i c f o c u s i n g ( F i g . 11, page 150), and by amino a c i d a n a l y s i s (Table XV, column B, page 136) f a i l e d to show any d i f f e r e n c e between them; and rechromatography of peak 1 on CM Sephadex showed that i t moved i d e n t i c a l l y to peak 2, when i t was a p p l i e d to the column i n low volume ( F i g . 12a, b, page 1 5 4 ) . This e v i -dence s t r o n g l y suggested that the two n e u t r a l f r a c t i o n s were i d e n t i c a l i n t h e i r neurohypophysial p e p t i d e c o n t e n t s , and they were combined f o r f u r t h e r a n a l y s i s . The s p l i t t i n g of the o x y t o c i c p r i n c i p l e on t h i s i o n exchanger may have r e -s u l t e d from the l a r g e volumes i n which the samples were a p p l i e d during the p r e p a r a t i v e p u r i f i c a t i o n procedures. 2. b. The n e u t r a l f r a c t i o n appeared pharmacologi-c a l l y d i f f e r e n t from g l u m i t o c i n , the p r i n c i p l e found i n the skates (Table XIX, page 157). This was important because both the Squalus p r i n c i p l e and the p r i n c i p l e found i n most skates had appeared to be of the EOP I type; the pharmacolog-i c a l disagreement between the Squalus p r i n c i p l e and g l u m i t o c i n suggested that g l u m i t o c i n was not the n e u t r a l p r i n c i p l e found i n a l l elasmobranchs. 2. c. The amino a c i d analyses of the p u r i f i e d n e u t r a l f r a c t i o n (Table XX, page 160) were the f i r s t analyses to be made of the o x y t o c i c p r i n c i p l e of a shark. They r e v e a l e d more amino a c i d r e s i d u e s than would be expected f o r a s i n g l e 167 neurohypophysial p e p t i d e , but which could represent a mixture of two such p r i n c i p l e s , present i n equimolar amounts and having an average a c t i v i t y of 109 IU/mg. Chapter k DISCUSSION I. STUDIES OF THE NEUROHYPOPHYSIAL PRINCIPLES OF THE SKATE, RAIA RHINA 1• The V a r i a b i l i t y i n the Apparent B i o l o g i c a l  Potency of Crude E x t r a c t s and of Gl u m i t o c i n The unexpected v a r i a t i o n which was found i n the mag-nesium p o t e n t i a t i o n values f o r neurointermediate lobe e x t r a c t s of Raia r h i n a (ex. R = 2.5 to l 6 . 8 ; see Table V, page 79) r e f l e c t e d changes i n . t h e apparent potency of the e x t r a c t s as judged by e i t h e r of the two assays u s e d — r a t uterus with magnesium i o n s , or r a t uterus i n the absence of magnesium (Chapter 3, S e c t i o n IB2 , page 71). The causes of the v a r i a t i o n i n potency are unknown, since the mechanism of the hormones' a c t i o n on the uterus and the r o l e p l a y e d by magnesium i n t h i s process are u n c e r t a i n (Munsick and Jeronimus, I965; B e n t l e y , 1965 ; Walter e t a l . , 1968; Krejcfi and Polacek , 1968). The potency of a neuro-hypophysial peptide other than o x y t o c i n i s determined by the absolute amount of the agent, and by the d i f f e r e n c e between the s e n s i t i v i t y of the assay p r e p a r a t i o n to the unknown peptide and to the o x y t o c i n standard. On each type of assay-r a t u t e r u s , m i l k - e j e c t i o n , e t c . - the d i f f e r e n c e i n the 168 169 responses of i n d i v i d u a l p r e p a r a t i o n s to o x y t o c i n and to i t s analogues has been r e l a t i v e l y constant and c h a r a c t e r i s t i c f o r each analogue, as evidenced by the apparent s t a b i l i t y of the b i o l o g i c a l p r o p e r t i e s which have appeared to c h a r a c t e r i z e each of the neurohypophysial p r i n c i p l e s (Perks and Dodd, 1963a; F o l l e t t and H e l l e r , 196Ua; 1964b; Sawyer, 1968). However, i t i s p o s s i b l e that some p r e p a r a t i o n s may make an anomalous d i s -t i n c t i o n between o x y t o c i n and the unknown, with a r e s u l t i n g v a r i a b i l i t y i n the potency of the analogue upon repeated assays. F u r t h e r , the p a r t i c u l a r d i f f e r e n c e i n s t r u c t u r e between o x y t o c i n and c e r t a i n of i t s analogues, such as the R_. r h i n a p r i n c i p l e , might promote such anomalous behavior i n the case of a p a r t i c u l a r p r i n c i p l e . This might be expected when the unknown p r i n c i p l e i s d e r i v e d from a c l a s s of animals which i s as widely removed from the mammals as the elasmo-branch f i sh . One f a c t o r which might i n f l u e n c e the behavior of the r a t uterus i s i t s stage i n the estrous c y c l e at the time of assay. Munsick and Jeronimus (1965) found that the potencies of some analogues, as measured both with and without Mg + +, were s i g n i f i c a n t l y a f f e c t e d by the estrous c o n d i t i o n of the u t e r i on which they were assayed. However the widest v a r i a -t i o n they found i n p o t e n t i a t i o n was only from 1.23 to I.98, values which were obtained from assays of a r g i n i n e v a s o t o c i n on u t e r i which were taken from r a t s i n m e t e s t r o u s - d i e s t r o u s and induced e s t r o u s , r e s p e c t i v e l y . In the s t u d i e s presented 170 here, the wide range of p o t e n t i a t i o n s was obtained on u t e r i a l l of which were judged to be i n the estrous s t a t e . How-ever, i n t h i s regard S t a p l e s and G e i l s (1965) have r e p o r t e d that approximately twelve hours before proestrous the r a t v a g i n a l smear c o n s i s t s of l a r g e c o r n i f i e d c e l l s , and these authors c a u t i o n that the e v a l u a t i o n of the smear i s not as s t r a i g h t - f o r w a r d as has been supposed. Nevertheless even i f some of the smears taken i n the present study were thus mis-i d e n t i f i e d , v a r i a t i o n of the magnitude observed by Munsick and Jeronimus could not account f o r the t o t a l v a r i a t i o n which was observed here. A l s o , i n t h i s r egard, Sawyer et a l . (1969) observed a range of magnesium p o t e n t i a t i o n s of from 1.U6 to 4.90 f o r s y n t h e t i c g l u m i t o c i n even when the u t e r i used f o r assay were taken from r a t s p r e t r e a t e d with d i e t h y l -s t i l b e s t r o l . T h e r e f o r e , while d i f f e r e n c e s i n the estrous c o n d i t i o n of the u t e r i may c o n t r i b u t e to the v a r i a b i l i t y i n magnesium p o t e n t i a t i o n , they are u n l i k e l y to be the sole cause. In a d d i t i o n to the v a r i a b i l i t y found i n magnesium p o t e n t i a t i o n s , some v a r i a b i l i t y was.also observed i n the m i l k - e j e c t i o n a c t i v i t i e s of the unknowns when they were assayed a g a i n s t o x y t o c i n . Two d i f f e r e n t p o t e n c i e s , the second being 100% g r e a t e r than the f i r s t , were found f o r both crude R. r h i n a e x t r a c t and f o r a s o l u t i o n of s y n t h e t i c g l u m i t o c i n when they were assayed together against s y n t h e t i c o x y t o c i n on two d i f f e r e n t occasions (see Table V I I I , page 88 ); n a t u r a l l y , the corresponding ratiosu of m i l k - e j e c t i o n to r a t uterus a c t i v i t y 171 (R,,„) r e f l e c t e d these d i f f e r e n c e s . ME Pharmacological a c t i v i t y r a t i o s have been used e x t e n s i v e l y to c h a r a c t e r i z e neurohypophysial p e p t i d e s . In p a r t i c u l a r , . v a l u e s f o r magnesium p o t e n t i a t i o n (R ) and r e l a t i v e m i l k - e j e c t i o n a c t i v i t y (R^-g) have been used to d i s -t i n g u i s h between the numerous p o s t u l a t e d elasmobranch p r i n c i p l e s (see Table I I I , page 33). In view of the obser-v a t i o n s presented here, i n which the v a r i a b i l i t y of these parameters has been demonstrated, i t i s c l e a r that they may be used to d i s t i n g u i s h neurohypophysial peptides from o x y t o c i n i t s e l f , i f o x y t o c i n i s used as the standard p r e p a r a t i o n , but that the values obtained by independent, " c l a s s i c a l " methods of assay a-r:e?anmn.r•e'1'i.a-Bl-.e c r i t e r i o n f o r comparing two unknowns - at l e a s t i n the case of elasmobranch e x t r a c t s . Consequently, some of the d i s t i n c t i o n s between the many elasmobranch p r i n c i -p l e s may prove to be i n v a l i d , and i t i s p o s s i b l e that the confusing array of these agents - EOP I, EOP I I , g l u m i t o c i n , the E^ p r i n c i p l e s , and the E^ p r i n c i p l e s - might be c o n s i d e r -ably s i m p l i f i e d , i n the f o l l o w i n g manner. The E^ and E^ p r i n c i p l e s (the c h r o m a t o g r a p h i c a l l y slow-moving and c h r o m a t o g r a p h i c a l l y fast-moving components, r e s p e c t i v e l y , of the o x y t o c i c a c t i v i t y found i n c e r t a i n s p e c i e s ) have been found to vary in. t h e i r magnesium p o t e n t i a -t i o n s ; three groups o f . v a l u e s have been observed f o r the R 1 i g of the slow-moving•component (R„ values of 6, 2, and l ) , and i i g two groups of values have been found f o r the fast-moving 172 component (R values of 2 and 1; see Table I I I , page 33). Therefore i t has been suggested ( H e l l e r and Roy, 1965b; Roy, 1969) that the nature of each of the two chromatographic components may vary between s p e c i e s . However, the t o t a l range of magnesium p o t e n t i a t i o n values shown by each f r a c t i o n (slow-moving f r a c t i o n : R = 0.95-6.3; fast-moving f r a c t i o n : i i g RMg = 0.85-2.6; see Table I I I ) i s c o n s i d e r a b l y smaller than the range of values shown e i t h e r by s y n t h e t i c g l u m i t o c i n (R = 1.46-9-5; see below) or by " n a t u r a l " g l u m i t o c i n as p o s t u l a t e d i n the Raia r h i n a e x t r a c t (R„, = 1.8-17.5. Table * Mg 1 ' » IV, page 73). The c a p r i c i o u s n e s s of t h i s r a t i o as i t a p p l i e s to elasmobranch p r i n c i p l e s s t r o n g l y suggests that the three groups of values found f o r E^ may a l l c h a r a c t e r i z e a s i n g l e slow-moving component and that the two groups of values found f o r E may a l s o c h a r a c t e r i z e a s i n g l e fast-moving agent. Other pharmacological evidence i s compatable with t h i s sugges-t i o n . The s l i g h t l y p o t e n t i a t e d E^ agent (R M g=2) and the more h i g h l y p o t e n t i a t e d E agent (R =6) appeared s i m i l a r i n t h e i r r e l a t i v e guinea p i g m i l k - e j e c t i o n a c t i v i t i e s and i n t h e i r r e l a t i v e r a t a n t i d i u r e t i c a c t i v i t i e s (Roy, 1969). The non-p o t e n t i a t e d E^ p r i n c i p l e was not assessed f o r these a c t i v i t i e s The non-potentiated E^ p r i n c i p l e d i d not d i f f e r from the p o t e n t i a t e d E^ agent i n i t s r e l a t i v e chicken blood pressure a c t i v i t y . However, the two p r i n c i p l e s , E^ and ~E , are s t i l l d i s t i n g u i s h e d by t h e i r d i f f e r e n t m o b i l i t i e s on paper chroma-tography (see Table I I I ) and by t h e i r d i f f e r e n t n a t r i f e r i c 173 a c t i v i t i e s , where t h i s l a t t e r c h a r a c t e r i s t i c has been i n v e s t i -gated (Roy, 1969). G l u m i t o c i n , c h e m i c a l l y i d e n t i f i e d i n s e v e r a l species of skate, has been d i s t i n g u i s h e d from the other elasmobranch n e u t r a l p r i n c i p l e s by i t s h i g h , t e n - f o l d , magnesium p o t e n t i a -t i o n (Acher e_t_ aJL. , 1965; see Table I I I ) ; a sample of s y n t h e t i c g l u m i t o c i n was found here to have a s i m i l a r l y high p o t e n t i a -t i o n (R = 9*5; Table V I I I , page 88 ). However, during the course of t h i s work, and i n a d d i t i o n to the o b s e r v a t i o n s made here, Sawyer et_ al_. (1969) found a lower range of values ^Mg = -'-•^6-4.90) f o r the same sample of s y n t h e t i c g l u m i t o c i n ; the d i f f e r e n t p o t e n t i a t i o n s observed f o r s y n t h e t i c g l u m i t o c i n i n the two l a b o r a t o r i e s (^j^g = 1.46-9.5) i n c l u d e d values which had been r e p o r t e d p r e v i o u s l y f o r EOP I (R = 1.2-3.2), CD E 0 P 1 1 ( RMg = 2 ' 6 ) ' E l ( HMg = °-95-6.3), and E 2 ( R M g = 0.85-2.6; see Table I I I ) . Therefore g l u m i t o c i n cannot now be shown to be d i f f e r e n t from any of the E 1 , E , EOP I, or EOP II p r i n c i p l e s on the b a s i s of i t s magnesium p o t e n t i a t i o n ; f u r t h e r , i n view of the range of R„ values that might be a t t r i b u t e d to each & Mg B of the s i n g l e E^ and E^ p r i n c i p l e s (see above), no d i s t i n c t i o n can r e l i a b l y be made between the magnesium p o t e n t i a t i o n s of any two of the p o s t u l a t e d elasmobranch n e u t r a l p r i n c i p l e s . However, de s p i t e doubts concerning the v a l i d i t y of the c r i -t e r i a which have been used to d i s t i n g u i s h the v a r i o u s elasmo-branch p r i n c i p l e s , i t i s f a i r to p o i n t out that even i n the case of agents which show no r e l i a b l e d i f f e r e n t i a t i o n by the 174 m e t h o d s u s e d , t h e c h e m i c a l n a t u r e may s t i l l p r o v e t o v a r y . No d i f f e r e n c e h a s y e t b e e n d e m o n s t r a t e d b e t w e e n E 2 a n d EOP I . T h e i r R ' s h a v e a p p e a r e d s i m i l a r a n d t h e y h a v e t h e same c h r o m a t o g r a p h i c m o b i l i t y i n n - b u t a n o l : a c e t i c a c i d : w a t e r = 4:1:5 ( s e e T a b l e I I I ) . O t h e r c o m p a r a t i v e d a t a i s l a c k i n g . E , EOP I , a n d g l u m i t o c i n h a v e a l l b e e n r e p o r t e d i n t h e same s p e c i e s , R a i a c l a v a t a ( H e l l e r a n d R o y , 1965a; P e r k s a n d D o d d , 1963a; A c h e r e_t , 1965)- T h i s s t r o n g l y s u g g e s t s t h a t t h e t h r e e a r e i d e n t i c a l , a l t h o u g h i t d o e s n o t p r o v e t h a t t h i s i s t h e c a s e . On t h e o t h e r h a n d , t h e a m i n o a c i d c o n t e n t o f g l u m i t o c i n i s i d e n t i c a l t o t h a t o f EOP I I , a n d a l t h o u g h t h e a m i n o a c i d s e q u e n c e c o u l d be d i f f e r e n t i n t h e t w o p e p t i d e s , i t i s a l s o p o s s i b l e t h a t i t c o u l d be t h e s a m e . The p a p e r c h r o m a t o g r a p h i c b e h a v i o r o f e i t h e r g l u m i t o c i n o r EOP I I h a s n o t b e e n d e t e r m i n e d a n d so i t c a n n o t be c o m p a r e d t o t h a t o f E , E , o r EOP I . The d i s t i n c t i o n b e t w e e n EOP I a n d EOP I I h a s b e e n b a s e d on t h e d i f f e r e n c e b e t w e e n t h e i r r e l a t i v e p o t e n c i e s on t h e m i l k - e j e c t i o n a s s a y (R ) . I n t h e s t u d y p r e s e n t e d h e r e , t h i s v a l u e h a s b e e n f o u n d t o v a r y i n t h e c a s e o f b o t h s y n -t h e t i c g l u m i t o c i n (R^-g = 8.2 a n d 15.6) a n d o f t h e R . r h i n a e x t r a c t (R^-g = 4 . 4 , 8.3, a n d 17-5). A n o t h e r v a l u e f o r t h e R o f s y n t h e t i c g l u m i t o c i n h a s b e e n r e p o r t e d b y S a w y e r e t a l . (1969) as 5.3. The v a l u e s o f R.,_, w h i c h c h a r a c t e r i z e t h e EOP I p r i n c i p l e r a n g e f r o m 1.5 t o 4.5 i n d i f f e r e n t s p e c i e s 175 (Perks and Dodd, 1963a; Sawyer, 1965; see Table I I I , page 33). The values f o r R ^ of the EOP II p r i n c i p l e are i n a higher range, given as 8.5 and 10.3 (Perks and Sawyer, 1965 ; see Table I I I ) . However, the R value f o r s y n t h e t i c g l u m i t o c i n ranges from 5-3 to 15.6 (Sawyer et_ a_l. , 1969; data presented h e r e ) , and t h i s range s t r e t c h e s almost down to the values f o r EOP I, and up beyond those f o r EOP I I . More important, " n a t u r a l " g l u m i t o c i n , as p o s t u l a t e d i n the R. r h i n a e x t r a c t (see S e c t i o n 2, below), gave a range of R from h.k to 17-5, M i l values which i n c l u d e r a t i o s a s c r i b e d to both EOP I and EOP I I . These data suggest that the R,,„ r a t i o i s an u n r e l i a b l e c r i -o t > ME t e r i o n f o r d i s t i n g u i s h i n g between EOP I and EOP I I , and further,.they ^ suggest that no d i s t i n c t i o n can be made between g l u m i t o c i n and e i t h e r EOP I or EOP II on t h i s b a s i s . It may be concluded that a c r i t i c a l assessment of the data a v a i l a b l e at t h i s time f a i l s to show a c l e a r d i s t i n c -t i o n between the p r i n c i p l e s EOP I, EOP I I , g l u m i t o c i n , and E^ , and suggests that the wide v a r i e t y of elasmobranch n e u t r a l p r i n c i p l e s may p o s s i b l y be reduced to only two - E , which i s c l e a r l y d i f f e r e n t i a t e d from E^ and EOP I by i t s chromatogra-phic behavior ( H e l l e r and P i c k e r i n g , 196l; H e l l e r and Roy, 1965a; 1965b; Roy, 1969) but which i s not always present (Perks, 1966; Sawyer, 1967; S w i a t k i e w i c z , 1968; data presented h e r e ) , and g l u m i t o c i n . However, the l i m i t e d c r i t e r i a a v a i l -able at present may f a i l to d i s t i n g u i s h a l l peptides which might e x i s t w i t h i n the p i t u i t a r i e s of the d i f f e r e n t s p e c i e s . 176 2. The I d e n t i t y of the Neurohypophysial P r i n c i p l e s  of Raia r h i n a Although the c l a s s i c a l methods of " b i o l o g i c a l assay have y i e l d e d much important i n f o r m a t i o n on neurohypophysial p r i n c i p l e s , t h i s study has shown that i n c o n s i s t e n c i e s may occur i n the values obtained, since some assay p r e p a r a t i o n s may make anomalous d i s t i n c t i o n s between the d i f f e r e n t chemical s t r u c t u r e s of the unknown and of the standard. However t h i s can be turned to advantage i n e s t a b l i s h i n g the i d e n t i t y of an unknown by comparing i t d i r e c t l y with s y n t h e t i c peptides other than o x y t o c i n (Syntocinon). Two i d e n t i c a l p e p t i d e s , assayed one against the other, w i l l have the same r e l a t i v e -a c t i v i t i e s on every assay p r e p a r a t i o n , and on each type of assay. I f the peptides are not i d e n t i c a l , however, the a c t i v i t y of the one r e l a t i v e to the other may w e l l vary between assays. The advent of s y n t h e t i c g l u m i t o c i n made i t p o s s i b l e f o r the skate p r i n c i p l e to be assayed against a molecule of p o s s i b l y i d e n t i c a l s t r u c t u r e , and the r e s u l t s of these d i r e c t assays suggested that g l u m i t o c i n i s i n f a c t the n e u t r a l p r i n c i p l e i n t h i s s p e c i es (see Chapter 3, S e c t i o n I B 3 , page 82). The amino a c i d analyses of the n e u t r a l p r i n c i p l e (Table IX, page 99) s t r o n g l y supported the pha r m a c o l o g i c a l evidence that the p e p t i d e . i s g l u m i t o c i n (see Chapter 3, S e c t i o n ICh, page 98). 177 The i d e n t i f i c a t i o n of the R. r h i n a p r i n c i p l e as g l u m i t o c i n , when i t had f i r s t appeared to correspond to the EOP I p r i n c i p l e , i s c o n s i s t e n t with the suggestion that the v a r i e t y of elasmobranch p r i n c i p l e s might p o s s i b l y be reduced to two - E and g l u m i t o c i n . However i t should be noted that the Squalus p r i n c i p l e , which had a l s o appeared to be EOP I, was found here to be n e i t h e r g l u m i t o c i n nor E . I t appeared to c o n s i s t of a mixture of two p e p t i d e s , n e i t h e r of which has been p r e v i o u s l y demonstrated. This w i l l be d i s c u s s e d i n l a t e r s e c t i o n s . A small amount of b a s i c p r i n c i p l e was separated away from the n e u t r a l peptide of Raia r h i n a . I t was not s u b j e c t e d to f u r t h e r study; however, i t was t e n t a t i v e l y suggested to be a r g i n i n e v a s o t o c i n on the b a s i s of the s i m i l a r i t y of i t s chromatographic behavior to that of the a r g i n i n e v a s o t o c i n found i n Squalus e x t r a c t s and on the u b i q u i t y of a r g i n i n e v a s o t o c i n as the b a s i c peptide i n non-mammalian v e r t e b r a t e s (Chapter 3; S e c t i o n I C3b). The presence of a r g i n i n e vaso-t o c i n i n the R_. r h i n a e x t r a c t would account f o r 9-5% of the t o t a l o x y t o c i c a c t i v i t y ( a r g i n i n e v a s o t o c i n : B, = 2.0; ADH Sawyer, 196l) but i t would account f o r only 0.7% of the t o t a l p eptide by weight i n a corresponding mixture of a r g i n i n e v a s o t o c i n and g l u m i t o c i n . 178 I I . STUDIES OF THE NEUROHYPOPHYSIAL PRINCIPLES OF THE DOGFISH, SQUALUS ACANTHIAS (PACIFIC VARIETY) 1. The I d e n t i t y of the Basic P r i n c i p l e of Squalus  acanthias The amount of b a s i c peptide present i n the e x t r a c t was r e l a t i v e l y very s m a l l , approximately 2% of the t o t a l o x y t o c i c a c t i v i t y which was e l u t e d from the CM Sephadex. It resembled a r g i n i n e v a s o t o c i n both c h r o m a t o g r a p h i c a l l y ( F i g . 8, page 128) and p h a r m a c o l o g i c a l l y (Table XVI A, page 138). A r g i n i n e v a s o t o c i n i s notable f o r the remarkably high and c h a r a c t e r i s t i c value of i t s r e l a t i v e f r o g bladder, a c t i v i t y . The Squalus p r i n c i p l e showed a s i m i l a r l y high a c t i v i t y on the f r o g bladder when the assays were performed against the usual o x y t o c i n standard (Syntocinon). This evidence confirmed Sawyer's ob s e r v a t i o n s on the same species (Sawyer, 1967). The i d e n t i t y of the Squalus b a s i c peptide with a r g i n i n e v a s o t o c i n was f u r t h e r s u b s t a n t i -ated by the more s o p h i s t i c a t e d technique of d i r e c t pharmaco-l o g i c a l comparison with the s y n t h e t i c peptide (Table XVI B), a method which had not been c a r r i e d out p r e v i o u s l y . The a c t i v i t y of the Squalus p r i n c i p l e r e l a t i v e to s y n t h e t i c a r g i -nine v a s o t o c i n was constant on the r a t u t e r u s , both i n the presence and absence of magnesium, on the a n t i d i u r e t i c assay, and on the f r o g - b l a d d e r assay. These - r e s u i t s i n d i c a t e t h at the Squalus b a s i c neurohypophysial peptide i s almost c e r t a i n l y i d e n t i c a l to a r g i n i n e v a s o t o c i n . However, chemical c o n f i r -mation i s s t i l l r e q u i r e d f o r complete c e r t a i n t y . 179 A r g i n i n e v a s o t o c i n i s the b a s i c peptide e l a b o r a t e d by other non-mammalian v e r t e b r a t e s , u s u a l l y i n higher p r o-p o r t i o n s than are found i n elasmobranchs. In Squalus the presence of a r g i n i n e v a s o t o c i n would account f o r only 2.3% of the t o t a l neurohypophysial a c t i v i t y ( r a t uterus a c t i v i t y ) , and only 2.k% of the t o t a l weight of neurohypophysial pep-t i d e p r e s e n t . 2. C o n s i d e r a t i o n s of the. I d e n t i t y of the N e u t r a l  P r i n c i p l e of Squalus acanthias a. Behavior of the n e u t r a l f r a c t i o n on CM  Sephadex The nature of the Squalus n e u t r a l p r i n c i p l e i s more e l u s i v e . I t was e l u t e d from CM Sephadex i n two f r a c t i o n s ( F i g . 91 12a; page 133, 154). However these two f r a c t i o n s were i n d i s t i n g u i s h a b l e from one another by any method that was attempted (see Chapter 3, S e c t i o n I I C 7 , page I 4 l ) . The apparent s i m i l a r i t y of the p r i n c i p l e i n each of the two f r a c t i o n s was s u b s t a n t i a t e d when f r a c t i o n 1 ( P r e p a r a t i o n h) was r e a p p l i e d to a CM Sephadex column i n a much smaller volume than had been used i n the f i r s t p u r i f i -c a t i o n (100 ml i n s t e a d of 2485 ml); subsequently, i t e l u t e d i n the same p o s i t i o n as i t s s i s t e r peak, f r a c t i o n 2 ( F i g . 12b; page 1 5 4 ) . F u r t h e r , i t could be shown that chromatography of a s i n g l e p e p t i d e , a p p l i e d to the column i n l a r g e volume, r e s u l t e d i n the p r o d u c t i o n of two a r t i f a c t peaks. This was demonstrated when the n e u t r a l f r a c t i o n of Raia. r h i n a , postu-180 l a t e d to be the s i n g l e p r i n c i p l e g l u m i t o c i n , was a l s o s p l i t i n t o two a c t i v e peaks by the o r i g i n a l procedure. Because of these c o n s i d e r a t i o n s , the two n e u t r a l f r a c t i o n s separated from Squalus e x t r a c t s ( P r e p a r a t i o n h) by the use of CM Sephadex were combined f o r subsequent study; t h i s allowed s u f f i c i e n t m a t e r i a l f o r the p o s s i b i l i t y of chemical a n a l y s i s . b. Chromatographic and p harmacological charac- t e r i s t i c s of the n e u t r a l p r i n c i p l e The p harmacological a c t i v i t y r a t i o s of the crude e x t r a c t s (Table X, page 106) and of the p u r i f i e d n e u t r a l f r a c t i o n (Table XVII, page 1^5 ) suggested the n e u t r a l p r i n c i -p l e i s an EOP I type of elasmobranch peptide which, as d i s c u s s e d i n the previous s e c t i o n ( S e c t i o n I , above) i s not c l e a r l y d i s t i n g u i s h e d from g l u m i t o c i n and might p o s s i b l y be i d e n t i c a l to i t . However the d i r e c t pharmacological compari-son of the p u r i f i e d agent against s y n t h e t i c g l u m i t o c i n (Table XIX, page 157) showed that the two p r i n c i p l e s were d i f f e r e n t . This confirmed the r e s u l t s of Sqwyer et_ a l . (1969) on. the same s p e c i e s , A t l a n t i c v a r i e t y , and i t i s important because i t suggests that g l u m i t o c i n i s not a n e u t r a l p r i n c i p l e common to a l l elasmobranchs. A f i r s t a l t e r n a t i v e to the i d e n t i f i c a t i o n of the Squalus n e u t r a l p r i n c i p l e with g l u m i t o c i n , i s that the unknown may be a mixture of the two p r i n c i p l e s , E^ and E^ , as found by H e l l e r and h i s co-workers i n many elasmobranch s p e c i e s . E^ and E^ have been d i s t i n g u i s h e d by t h e i r d i f f e r e n t m o b i l i t i e s 181 on paper chromatograms (developed i n n-butanol : a c e t i c a e i d : water = 4:1:5); i n some species they have a l s o d i f f e r e d i n t h e i r magnesium p o t e n t i a t i o n s , so f a r as t h i s c r i t e r i o n i s r e l i a b l e (see Table I I I , page 33). However, there was no evidence f o r the presence of the c h r o m a t o g r a p h i c a l l y slow-moving component, E , i n the P a c i f i c v a r i e t y of.Squalus  ac a n t h i as, as the f o l l o w i n g evidence w i l l show: 1. Paper chromatograms o f : a) Squalus crude e x t r a c t ( F i g . 5b, page 115), b) Squalus neurohypophysial m a t e r i a l par-t i a l l y p u r i f i e d by f i l t r a t i o n through Sephadex G-15 g e l ( F i g . 5<i) , and c) the Squalus n e u t r a l f r a c t i o n a f t e r e l u t i o n from CM Sephadex - both the f r a c t i o n 1 component ( F i g . 5e) and the f r a c t i o n 2 component ( F i g . 5f) - were developed i n n - b u t a n o l : a c e t i c acid:water = 4:1:5. A l l showed only one area of o x y t o c i c a c t i v i t y , from R„ 0.4 to 0.7, the r p o s i t i o n c h a r a c t e r i s t i c of EOP I and of E^. The chromatograms w i l l be dis.cussed i n t u r n : a. The glands had been e x t r a c t e d at a high c o n c e n t r a t i o n (40 mg d r i e d t i s s u e / m l ) and i t was p o s s i b l e that the p r o t e i n content of the crude e x t r a c t prevented a c l e a r r e s o l u t i o n of E^ from E^ on the chromatogram. However, there was no evidence f o r the presence of E^ i n the t a i l i n g edge of the a c t i v e peak, as judged by the magnesium p o t e n t i a -182 t i o n s of the e l u a t e s (see Chapter 3, S e c t i o n IIB2). Although magnesium p o t e n t i a t i o n values have been shown to be an u n r e l i -able c r i t e r i o n f o r comparing elasmobranch agents, i t i s s t i l l necessary to use these r e s u l t s i n d i s c u s s i o n i n order to r e l a t e the present f i n d i n g s to work p r e v i o u s l y p u b l i s h e d . b. The amount of Sephadex G - 1 5 - p u r i f i e d m a t e r i a l loaded onto the chromatogram was high (106.7 mU) , and i n t h i s case only, a small amount of o x y t o c i c a c t i v i t y (1% of the t o t a l ) was found i n the e l u a t e of R 0..3-0.4. This a c t i v i t y had the pharmacological c h a r a c t e r i s t i c s of a r g i n i n e v a s o t o c i n (Table XVIA, page 138). E^ had been r e p o r t e d i n a s i m i l a r p o s i t i o n , R 0.35-0 .45 , on a paper chromatogram of Squalus e x t r a c t ( H e l l e r and P i c k e r i n g , 1 9 6 l ) . In the present case, i t i s u n l i k e l y that the small amount of a r g i n i n e v a s o t o c i n p o s t u l a t e d i n R„ 0.3-0 .4 could have masked the presence of E i n the same e l u a t e , f o r the f o l l o w i n g reasons: i . The a c t i v i t y i n R_, 0.3-0 .4 accounted r + + f o r only 1% of the t o t a l r a t uterus a c t i v i t y (no Mg present) recovered from the chromatogram, and E^ i s . r e p o r t e d to occur i n much l a r g e r amounts - from one t h i r d of the t o t a l a c t i v i t y ( P i c k e r i n g , p e r s o n a l communication to Perks, i960) to one h a l f of the t o t a l a c t i v i t y (Roy, 1969). i i . The presence of E^ as a high pro-p o r t i o n of the t o t a l a c t i v i t y (one t h i r d to one h a l f ) would 183 have "been expected to lower the r a t i o s R and R._„ of the r B ADH e l u a t e of the slow-running component (R„ 0.3-O .U) to values r below those of a r g i n i n e v a s o t o c i n . T h i s was not the case (Table XVIA, page 138). i i i . The slow-moving E^ agent has been re p o r t e d i n p o s i t i o n s as low as Rp 0.04 upon paper chromato-graphy of neurohypophysial m a t e r i a l from other elasmobranch species (ex. Raia c l a v a t a ; H e l l e r and Roy, 1965a). No a c t i v i t y was detected i n R^ , 0-0.3 of the Squalus chromatogram. While the E n p r i n c i p l e might have been co n f i n e d to one R_ 1 r u n i t , R,-, 0.3-0.4, t h i s would be somewhat s u r p r i s i n g i n view r of the s i z e of the l o a d which was a p p l i e d to the chromatogram and the p r o p o r t i o n i n which E^ i s r e p o r t e d to occur. i v . Paper chromatograms of the n e u t r a l p r i n c i p l e , a f t e r i t had been separated from the a r g i n i n e v a s o t o c i n on CM Sephadex, showed no a c t i v i t y i n R„ 0.3-0.4. 2. It should be noted that the two n e u t r a l f r a c t i o n s which were e l u t e d from CM Sephadex d i d not c o r r e s -pond to the E^ and E^ p r i n c i p l e s . In f a c t , they were i n d i s -t i n g u i s h a b l e , and n e i t h e r had the high magnesium p o t e n t i a t i o n sometimes c h a r a c t e r i s t i c of "E^ (Table XVII, page 145), nor e x h i b i t e d i t s slow-running chromatographic behavior ( F i g . 5, e and f ; page 115). 3. There i s the p o s s i b i l i t y that the E 18U p r i n c i p l e of Squalus might have been l o s t i n the p r o c e s s i n g of the glands or of the e x t r a c t . This i s u n l i k e l y , f o r the f o l l o w i n g reasons : a. Only about k% of the t o t a l o x y t o c i c a c t i v i t y ( r a t uterus a c t i v i t y , without Mg ) of the glands was l o s t i n t o the acetone i n which they were c o l l e c t e d (Table XI, page n o ) . F u r t h e r , t h i s a c t i v i t y d i d not d i f f e r , by the c r i t e r i a which d i s t i n g u i s h e s from E , from the m a t e r i a l which was l a t e r e x t r a c t e d from the glands by a c e t i c a c i d . I t resembled the crude a c e t i c a c i d e x t r a c t i n i t s magnesium p o t e n t i a t i o n value of approximately 2 and i n i t s f a s t - r u n n i n g behavior upon paper chromatography i n n - b u t a n o l : a c e t i c a c i d : water = h : 1 :5 (Table X I I , page 112; F i g . 5, b and c, page 115). In c o n t r a s t , E i s c h r o m a t o g r a p h i c a l l y slow-running and i n some species i t may be d i s t i n g u i s h e d from the E^ p r i n c i p l e by i t s higher R values (Table I I I , page 33). There was no evidence f o r an incomplete s e p a r a t i o n of E^ from E on the chromatogram of the acetone s o l u b l e f r a c t i o n , as judged by the magnesium p o t e n t i a t i o n of the e l u a t e s , so f a r as t h i s i s admissable evidence (see Chapter 3, S e c t i o n II B2). b. During the passage of the e x t r a c t through Sephadex G-15, approximately l.h% of the o x y t o c i c a c t i v i t y was e l u t e d with the Lowry peptide peak i n advance of the main o x y t o c i c peak ( F i g . 7» page 123). It i s u n l i k e l y t h at t h i s m a t e r i a l could account f o r the "missing" E p r i n c i p l e s i n c e 185 E^ i s r e p o r t e d to occur i n amounts approximately equal to those of the E^ p r i n c i p l e ( P i c k e r i n g , p e r s o n a l communication to Perks, I960; Roy, 1969). A l s o , i t i s u n l i k e l y t h at a mixture of two n e u t r a l neurohypophysial p r i n c i p l e s such as E^ and E^ would have been r e s o l v e d on Sephadex G-15 when, as demonstrated by subsequent paper chromatography and ion exchange chromatography of the main el u a t e peak, the b a s i c p e p t i d e , a r g i n i n e v a s o t o c i n , had remained i n a s s o c i a t i o n with the n e u t r a l f r a c t i o n throughout the g e l f i l t r a t i o n procedure ( F i g s . 8 and 9> pages 128 and 133) . c. Amino a c i d analyses of the n e u t r a l f r a c t i o n . The pharmacological and chromatographic c h a r a c t e r -i s t i c s of the Squalus n e u t r a l p r i n c i p l e demonstrated that i t i s n e i t h e r o x y t o c i n , g l u m i t o c i n , nor a mixture of the E^ and Eg p r i n c i p l e s . However, the amino a c i d analyses of the p u r i f i e d m a t e r i a l (Table XX) do suggest that i t may be a mixture of two neurohypophysial p e p t i d e s , present i n equal amounts and having the high average potency of 109 IU/mg. It i s not l i k e l y t h a t a l l the amino acids i n the h y d r o l y s a t e would have occurred together i n one very long peptide. Such a s t r u c t u r e has not been found among n a t u r a l l y o c c u r r i n g neurohypophysial hormones. Furthermore, s t u d i e s of s y n t h e t i c p eptides with s l i g h t l y elongated molecules show that a d d i t i o n a l amino a c i d r e s i d u e s i n the chain lower the potency (Berde and Boissonnas, 1966), and the a c t i v i t y of 186 the Squalus n e u t r a l f r a c t i o n i s high. It i s p o s s i b l e that the amino a c i d analyses r e f l e c t e d only one neurohypophysial p e p t i d e , together with another contaminating peptide or with f r e e amino acids which had not been separated away during the p u r i f i c a t i o n . However, the f o l l o w i n g evidence i s s t r o n g l y i n favour of a "pure" prepa-r a t i o n of a peptide mixture: 1. The a n a l y s i s of an unhydrolyzed sample of p u r i f i e d m a t e r i a l r e v e a l e d only small amounts of two f r e e amino acids - s e r i n e and g l y c i n e (Table /XXI,pagel6l) - and the a n a l y s i s of the corresponding h y d r o l y z e d sample (Table XX'.,;' column 2, pageLfjO1) has been c o r r e c t e d f o r these two res idues. 2. The HC1 used i n the hydrolyses was a l s o analyzed f o r f r e e amino a c i d s , and- only minimal amounts were detected (Table XXII, page ' l 6 3 ) . 3- The amino a c i d s i n the h y d r o l y s a t e s were present i n a molar r a t i o to glutamic a c i d of 2:1 or 1:1. Although not i m p o s s i b l e , i t would be a notable c o i n c i d e n c e i f the free amino a c i d s , or acids due to another non-active p e p t i d e , happened to be present i n such p r e c i s e amounts. k. In t h i s and other l a b o r a t o r i e s , p u r i f i c a -t i o n procedures s i m i l a r to those followed here have been used s u c c e s s f u l l y to produce e f f e c t i v e l y pure neurohypophysial . 187 p r e p a r a t i o n s . In f a c t , the present procedure i n c l u d e d one more step - the f i n a l paper p a r t i t i o n chromatography - than was r e q u i r e d to prepare pure i s o t o c i n from salmon (Wilson and Smith, 1969), and two more steps - chromatography on SE Sephadex and on paper - than were needed to i s o l a t e g l u m i t o c i n from another elasmobranch, Raia o c e l l a t a (Perks and Sawyer, 1965 ; a l s o see Sawyer et_ al_. , 1969). 5. The a n a l y s i s of the n e u t r a l f r a c t i o n from P e r p a r a t i o n k a f t e r i t s e l u t i o n from the f i n a l paper chroma-togram (Table XX, column 3, page 160 ). was s i m i l a r to the a n a l y s i s made a f t e r the preceeding chromatography on SE Sephadex ( Table XX- column 2). I t would be most s u r p r i s i n g i f a non-neurohypophysial contaminating peptide showed both the same e l e c t r o s t a t i c behavior as the neurohypophysial p r i n c i p l e upon ion exchange chromatography at two d i f f e r e n t pH's - 5.0 and 2.U5 -, and a l s o the same r e l a t i v e s o l u b i l i t y i n the sol v e n t system used f o r the p a r t i t i o n chromatography on paper. 6. The average s p e c i f i c a c t i v i t y of the pep-t i d e mixture i s 109 IU/mg. This i s i n the range of a c t i v i t y of two other n a t u r a l l y o c c u r r i n g n e u t r a l analogues of ox y t o c i n - mesotocin, 290 IU/mg. and i s o t o c i n , 150 IU/mg (Sawyer, 1965) - a n G ^ i s much higher than the a c t i v i t y of g l u m i t o c i n , 8 IU/mg (Table y l H , page 88-). I t i s a l s o w i t h i n the range o f , or higher than, the a c t i v i t y of the many 188 s y n t h e t i c analogues which have been s t u d i e d , with the exception of des amino-oxytocin (Sawyer, 1965 ; Chan e_t a l . , 1963). This high average a c t i v i t y speaks f o r a "pure" p r e p a r a t i o n ; i t a l s o i n d i c a t e s that i f g l u m i t o c i n were i t s e l f p r e s e n t , i t woul'dbe mixed with a f u r t h e r peptide of remark-ably high a c t i v i t y . 7. Recently Sawyer (personal communication) has r e s o l v e d two d i f f e r e n t samples of the p u r i f i e d Squalus n e u t r a l f r a c t i o n i n t o two components us i n g p a r t i t i o n chromatography on Sephadex columns. One of the samples was prepared i n Sawyer's l a b o r a t o r y with neurointermediate lobes taken from S qualus ac a n t h i as ( A t l a n t i c v a r i e t y ) caught o f f the coast of V i r g i n i a , and the other, S qualus ac anthi as ( P a c i f i c v a r i e t y ) , was p u r i f i e d i n t h i s l a b o r a t o r y , a part of P r e p a r a t i o n 1. The s e p a r a t i o n s were achieved i n three d i f f e r e n t s o l v e n t systems: 1) 1-butanol:1.5% p y r i d i n e i n 3.5% a c e t i c a c i d = 1.1 2) 1-butanol:ethanol:1.5% p y r i d i n e i n 3.5% a c e t i c a c i d = 7:2:9 3) 1-butanol:ethanol:1.5% p y r i d i n e i n 3.5% a c e t i c a c i d = 17:1:18 Whether or not these two components correspond to the two peptides i n the mixture r e p o r t e d here has not been e s t a b l i s h e d . However the independent r e s o l u t i o n of the n e u t r a l f r a c t i o n of P r e p a r a t i o n 1 i n t o two components by column p a r t i t i o n 189 chromatography prov i d e s powerful support f o r the e x i s t e n c e of a peptide mixture, as suggested here. It i s s u r p r i s i n g that the powerful e l e c t r o f o c u s i n g technique d i d not achieve a r e s o l u t i o n of the two p o s s i b l e p e p t i d e s . It i s p o s s i b l e that the two have i d e n t i c a l i s o -e l e c t r i c p o i n t s . Another e x p l a n a t i o n i s that one peptide i s l e s s a c t i v e than the other. T h i s would give r i s e to two p o s s i b i l i t i e s . F i r s t l y , while there was no evidence of "notching" i n the o x y t o c i c peak ( F i g . 11, page^^o), i t i s s t i l l p o s s i b l e t h a t there was a p a r t i a l r e s o l u t i o n of two p r i n c i p l e s of unequal a c t i v i t y . Secondly, the re c o v e r y of o x y t o c i c a c t i v i t y from the experiment suggested that i f the r a t i o of the s p e c i f i c a c t i v i t i e s of the two peptides (which amino a c i d a n a l y s i s suggested were equimolar) were i n the order of 10:1, the l e s s a c t i v e p r i n c i p l e , which may have been w e l l separated from the more a c t i v e one, would have escaped d e t e c t i o n on the r a t uterus assay. There have been other r e p o r t s of a mixture of n e u t r a l peptides o c c u r r i n g w i t h i n a s i n g l e s p e c i e s . For example, a n a l y s i s of the n e u t r a l f r a c t i o n from the cobra, Naj a naj a, has r e v e a l e d the amino a c i d s of an equimolar mixture of o x y t o c i n and mesotocin, and i t s b i o l o g i c a l a c t i v i t y r a t i o s support t h i s ( P i c k e r i n g , 1967). Pharmacological a c t i v i t y r a t i o s suggest that other r e p t i l e s , the grass snake, green t u r t l e , and Loggerhead t u r t l e , may a l s o e l a b o r a t e an e q u i -molar mixture of o x y t o c i n and mesotocin ( F o l l e t t , 1967). . 190 Within c e r t a i n elasmobranch species , i n c l u d i n g Squalus  a c a n t h i a s , two n e u t r a l p r i n c i p l e s and E^ have been r e p o r t e d ( H e l l e r and P i c k e r i n g , 1 96l; H e l l e r and Roy, 1965a; 1965b; 1967; Roy, 1969), although others (Perks, 1966; Sawyer, 1967; Swi a t k i e w i c z , 1968; data presented here) have been unable to repeat t h i s chromatographic r e s o l u t i o n , e i t h e r i n the case of Squalus or of other elasmobranch s p e c i e s . I t was demonstrated above that the peptide mixture suggested here does not correspond to a mixture of E^ and Eg A l s o , i t has already been noted that the two peptides suggested by the amino a c i d analyses do not correspond i n d i v i d u a l l y to the two n e u t r a l peaks which were e l u t e d o f f the CM Sephadex. d. T h e o r e t i c a l c o n s i d e r a t i o n s and s p e c u l a t i o n s  of p o s s i b l e s t r u c t u r e At t h i s time the amino a c i d content and molecular s t r u c t u r e of each of the two p o s s i b l e peptides can only be i n f e r r e d , and the f o l l o w i n g must be regarded as s p e c u l a t i o n . Taking the amino a c i d sequence of the known n a t u r a l l y occurring p e p t i d e s as a guide, the d u p l i c a t e amino a c i d r e s i d u e s found i n the Squalus n e u t r a l f r a c t i o n can be arranged i n t o two peptides with the f o l l o w i n g s t r u c t u r e : 1 2 3 * + 5 6 7 8 9 c y s - t y r asp (NH 2 ) - cys-pro gly(NH2) Since the o x y t o c i c a c t i v i t y of Squalus n e u r o i n t e r -mediate lobe e x t r a c t i s e n t i r e l y destroyed by i n c u b a t i o n 191 with 0.01 M sodium t h i o g l y c o l l a t e (Perks and Dodd, 1963b; Swiatkiewicz , 1968), the normal c y s t i n e r i n g i s assumed to be present i n both p e p t i d e s . P o s i t i o n s 3, 4 , and 8 would then remain u n f i l l e d , and i t i s i n these p o s i t i o n s that amino a c i d s u b s t i t u t i o n s have taken p l a c e i n the n a t u r a l l y o c c u r r i n g analogues of o x y t o c i n . The s i x remaining r e s i d u e s , s e r i n e , glutamine, a l a n i n e , v a l i n e , l e u c i n e , and i s o l e u c i n e , would be d i s t r i b u t e d among these s i x unoccupied p o s i t i o n s . G l u m i t o c i n , already known to be an elasmobranch pep-t i d e , might be considered as a f i r s t p o s s i b i l i t y f o r one of the Squalus p r i n c i p l e s . The necessary amino acids f o r the s t r u c t u r e 3 - i s o l e u c i n e , 4 - s e r i n e , and 8-glutamine were pre-sent i n the a n a l y s i s . However there i s the f o l l o w i n g phar-m a c o l o g i c a l and chromatographic evidence against the presence of g l u m i t o c i n . G l u m i t o c i n has a low a c t i v i t y of approximately 10 IU/mg and an R^ -g = 0.8 ± 0.1 (Sawyer, p e r s o n a l communi-c a t i o n , 1968). Therefore i t s companion peptide ( i f present i n equimolar amount, as i n d i c a t e d by the amino a c i d analyses) must have an a c t i v i t y of approximately 208 IU/mg and an R-p-g of at l e a s t 7 f o r the average Squalus a c t i v i t y of 109 IU/mg and even the minimum R^ -g value observed f o r the n e u t r a l f r a c t i o n (R-p-g = 6.9; Table XVII,page 1U5) to be r e a l i z e d . S i m i l a r high R F B values f o r the n e u t r a l f r a c t i o n of Squalus have been r e p o r t e d p r e v i o u s l y (R Fg = 3.0 or 4.2; Sawyer, 1965 ; 1967). With the exception of a r g i n i n e v a s o p r e s s i n , a l l the o x y t o c i n analogues with an R value g r e a t e r than one have an i n t a c t 192 o x y t o c i n r i n g , and those with an R__ of l e s s than one do not do so (Sawyer et_ al_. , 1 9 6 l ; 1970) . Therefore an i n t a c t oxy-t o c i n r i n g would he expected i n the second p e p t i d e . However the presence of both g l u m i t o c i n ( 3 - i s o l e u c i n e , ^ - s e r i n e , 8-glutamine) and a second peptide with the o x y t o c i n r i n g s t r u c -ture ( 3 - i s o l e u c i n e , 4-glutamine, 8 - a l a n i n e / v a l i n e / l e u c i n e ) i s incompatable with the Squalus amino a c i d content. More d i r e c t evidence against the i d e n t i t y of one of the Squalus components with g l u m i t o c i n comes from p a r t i t i o n chromatography on Sephadex columns. Sawyer (personal com-munication) has shown that g l u m i t o c i n runs with a d i f f e r e n t Rp from the two Squalus components i n the three solvent sys-tems l i s t e d above (page 188 ) . Three other n a t u r a l l y o c c u r r i n g n e u t r a l p e p t i d e s , o x y t o c i n , mesotocin, and i s o t o c i n , remain as p o s s i b i l i t i e s . Mesotocin and i s o t o c i n can be e l i m i n a t e d immediately. Each possesses a d u p l i c a t e i s o l e u c i n e residue and the Squalus a n a l y s i s y i e l d e d only one. Oxytocin i s a l s o an u l i k e l y candidate. I t s a c t i v i t y of U50 IU/mg would-mean- an average Squalus a c t i v i t y of at l e a s t 225 IU/mg, even i f the second peptide were almost i n e r t ; the observed value was only 109 IU/mg. A l s o , since the Squalus n e u t r a l f r a c t i o n showed high r e l a t i v e f r o g bladder a c t i v i t y (R =6.9/ and 17-2; see Table r B XVII, page 1U5), the value of R =1 f o r o x y t o c i n would r e q u i r e r B a second almost i n e r t peptide to have a very high R value and t h e r e f o r e an i n t a c t o x y t o c i n r i n g . The amino a c i d s of 193 t h e S q u a l u s n e u t r a l f r a c t i o n c a n n o t s a t i s f y t h e r e q u i r e m e n t s o f t w o s u c h r i n g s . F u r t h e r , S a w y e r (1967) h a s d e m o n s t r a t e d a d i f f e r e n c e b e t w e e n t h e s e t h r e e p e p t i d e s a n d t h e S q u a l u s n e u t r a l f r a c t i o n d u r i n g p a r t i t i o n c o l u m n c h r o m a t o g r a p h y i n 1 - b u t a n o l : b e n z e n e : p y r i d i n e : 0 . 1 % a c e t i c a c i d = 6:2:1:9» d e s p i t e t h e f a c t t h a t t h e S q u a l u s n e u t r a l f r a c t i o n m o v e d as a s i n g l e p e a k i n t h i s s o l v e n t s y s t e m . T h e r e f o r e i t a p p e a r s t h a t n e i t h e r o f t h e p o s s i b l e S q u a l u s p e p t i d e s h a s b e e n d e m o n s t r a t e d p r e v i o u s l y among t h e k n o w n n e u r o h y p o p h y s i a l h o r m o n e s . I n v i e w o f t h e h i g h S q u a l u s R v a l u e , one o f t h e p e p t i d e s m i g h t be a s s u m e d t o h a v e an i n t a c t o x y t o c i n r i n g ( S a w y e r e t a l . , 1961 ; 1970; S a w y e r , 19^5; 1967). The a m i n o a c i d s f o u n d i n S q u a l u s a l l o w f o r one o f a p o s s i b l e f o u r s u c h a n a l o g u e s , 8 - s e r i n e , 8 - v a l i n e , 8 - a l a n i n e , o r 8 - l e u c i n e o x y t o c i n . The 8 - l e u c i n e p e p t i d e i s o x y t o c i n i t s e l f , a n d as d i s c u s s e d a b o v e , i t i s p r o b a b l y n o t p r e s e n t . Of t h e t h r e e r e m a i n i n g a n a l o g u e s , a l l h a v e an a c t i v i t y o f a t l e a s t l U O I U / m g , an R v a l u e g r e a t e r r a t h a n o n e , a n d a l o w R . - . ^ v a l u e ( S a w y e r e t a l . , 1970). A i m " ~ — 8 - a l a n i n e o x y t o c i n , w i t h an a c t i v i t y o f a p p r o x i m a t e l y 15^ I U / m g a n d R_„ = 2 i s t h e l e a s t s a t i s f a c t o r y p o s s i b i l i t y s i n c e i t s a c c o m p a n y i n g p e p t i d e w o u l d be e x p e c t e d t o h a v e an a c t i v i t y o f 6h I U / m g a n d an R o f a t l e a s t 19» a n d a g a i n r a t h e a m i n o a c i d s f o u n d i n S q u a l u s do n o t p e r m i t t w o i n t a c t o x y t o c i c r i n g s . F u r t h e r m o r e , S a w y e r ( p e r s o n a l c o m m u n i c a t i o n ) 194 has shown t h a t 8 - a l a n i n e o x y t o c i n runs w i t h a d i f f e r e n t R F from both o f the Squalus components d u r i n g p a r t i t i o n column chromatography i n the t h r e e d i f f e r e n t s o l v e n t systems l i s t e d above (page 1 8 8 ) . P o s s i b l e v a r i a t i o n i n the s p e c i f i c a c t i v i t i e s and i n the r e l a t i v e f r o g b l a d d e r a c t i v i t i e s o f both the o x y t o c i n analogues and the Squalus n e u t r a l f r a c t i o n upon r e p e a t e d a s s a y s , makes u n c e r t a i n any s p e c u l a t i o n on the i d e n t i t y of the Squalus p e p t i d e s . T h e r e f o r e i t i s i m p o r t a n t t h a t t h e r e i s chromato-g r a p h i c e v i d e n c e as w e l l as a p o s s i b l e p h a r m a c o l o g i c a l b a s i s f o r the t h e o r e t i c a l e l i m i n a t i o n of these p e p t i d e s - o x y t o c i n , g l u m i t o c i n , i s o t o c i n , m e s o t o c i n , and 8 - a l a n i n e o x y t o c i n - from the Squalus p e p t i d e m i x t u r e . The r e m a i n i n g p o s s i b i l i t i e s f o r a p e p t i d e w i t h an i n t a c t o x y t o c i n r i n g are 8 - v a l i n e o x y t o c i n and 8 - s e r i n e o x y t o c i n . These two m o l e c u l e s have not been shown t o d i f f e r chromato-g r a p h i c a l l y from the Squalus n e u t r a l f r a c t i o n and so the p o s s i -b i l i t y o f t h e i r presence i n the Squalus p e p t i d e m i x t u r e can o n l y be c o n s i d e r e d on p h a r m a c o l o g i c a l grounds. The 8 - s e r i n e analogue has an a c t i v i t y of 145 IU/mg and an R of 9 (Sawyer r B et a l . , 1970). The companion p e p t i d e might then be e x p e c t e d t o have an R„_ of a p p r o x i m a t e l y 3 t o account f o r a l l the f r o g r B b l a d d e r a c t i v i t y of the Squalus n e u t r a l f r a c t i o n (R^-g = 6.9; Table X V I I , page 145). However the r e l a t i v e f r o g b l a d d e r a c t i v -i t y of the Squalus f r a c t i o n has v a r i e d on r e p e a t e d a s s a y s , 195 although i t s value f o r Rjpg has always appeared g r e a t e r than one. Rpg values of 6.9 and 1 7 « 2 were r e p o r t e d here (Table XVII) and lower values of 3.0 and 4 .2 have been r e p o r t e d elsewhere (Sawyer, 1965; 1967) . While the f r o g bladder a c t i v i t y of 8-serine o x y t o c i n might be i n s u f f i c i e n t to account f o r a l l the f r o g bladder a c t i v i t y i n an equimolar peptide mixture whose o v e r a l l Rpg was 6 . 9 , i t could account f o r the t o t a l f r o g bladder a c t i v i t y of a mixture whose value f o r R ^ was 3. In t h i s l a t t e r case the second peptide would be r e q u i r e d to con-t r i b u t e very l i t t l e f r o g bladder a c t i v i t y , and need not be presumed to possess an i n t a c t o x y t o c i n r i n g . The l a s t peptide to be considered i s 8 - v a l i n e o x y t o c i n . Two values have been r e p o r t e d f o r the s p e c i f i c a c t i v i t y of 8-v a l i n e o x y t o c i n - 200 IU/mg and 265 IU/mg (Sawyer et_ a l . , 1970) . The value of R F B t h i s peptide i s r e p o r t e d to be 6 (Sawyer et a l . , 1970) . I f the a c t i v i t y of the peptide were taken as the lower value of 200 IU/mg, then 8 - v a l i n e o x y t o c i n could account f o r a l l the f r o g bladder a c t i v i t y i n an equimolar mix-tu r e of peptides c h a r a c t e r i z e d by an R-pB of 3.0 or 4 . 2 , the lower values r e p o r t e d f o r the Squalus n e u t r a l f r a c t i o n (Sawyer, 1965 ; 1967); i t cou l d not account f o r a l l the f r o g bladder a c t i v i t y i f the R-p-g of the peptide mixture were 6 . 9 , the higher value f o r Squalus found here. The second peptide i n the mixture would r e q u i r e a high value f o r R-p-g of 17 (and an i n t a c t o x y t o c i n r i n g ) f o r an o v e r a l l R-p-g value of 6.9 to be 1 9 6 r e a l i z e d . I f , on the other hand, the a c t i v i t y of 8 - v a l i n e o x y t o c i n were taken to be the higher value of 2 6 5 IU/mg , the peptide could account f o r a l l the f r o g bladder a c t i v i t y i n a peptide mixture whose R was 6 . 9 ; i t could not account r B f o r a l l the f r o g bladder a c t i v i t y i n a mixture c h a r a c t e r i z e d by higher values f o r t h i s ratio:, and a value f o r R„„ of 1 7 . 2 was a l s o found here f o r the Squalus n e u t r a l f r a c t i o n . (The values f o r R of 6 . 9 and 1 7 . 2 were obtained f o r peak 1 and r B peak 2 of the n e u t r a l f r a c t i o n , r e s p e c t i v e l y ; the neurohypo-p h y s i a l peptide content of the two peaks was considered i d e n t i c a l and so the two values of R_^ are each c o n s i d e r e d to FB represent the e n t i r e n e u t r a l f r a c t i o n ) . On the b a s i s of the data a v a i l a b l e at the present time, i t must be concluded that there i s no c l e a r pharmacological evidence f o r the e l i m i n a t i o n of e i t h e r 8 - v a l i n e o x y t o c i n or 8 - s e r i n e o x y t o c i n from the p o s t u l a t e d Squalus peptide mixture. The l i m i t e d chromatographic evidence i s a l s o i n c o n -c l u s i v e . On p a r t i t i o n column chromatography i n 1 - b u t a n o l : e t h a n o l : 1 . 5 $ p y r i d i n e i n 3.5% a c e t i c a c i d = 1 7 : 1 : 1 8 , 8 - v a l i n e o x y t o c i n runs with the same R_ as one of the peaks of the Squalus n e u t r a l f r a c t i o n (Sawyer, p e r s o n a l communication). However not too' much emphasis should be p l a c e d on t h i s . In the same so l v e n t system o x y t o c i n runs with the same R as the other Squalus peak, and o x y t o c i n i s not con s i d e r e d to be a component of the' Squalus mixture. Both 8 - v a l i n e and 8 - s e r i n e o x y t o c i n are f e a s i b l e 197 elasmobranch peptides i n terms of the ge n e t i c code. The most widespread of the neurohypophysial hormones i s a r g i n i n e v a s o t o c i n which occurs i n a l l v e r t e b r a t e c l a s s e s and i s the only one so f a r known to be e l a b o r a t e d by the cyclostomes. Because of t h i s i t i s con s i d e r e d the most p r i m i t i v e of the pep-t i d e s , the one which gave r i s e through mutation to the o t h e r s , which are of more l i m i t e d occurrence. According to the genetic code as presented by V l i e g e n t h a r t and V e r s t e i g (1967), 8-serine o x y t o c i n c o u l d have developed from a r g i n i n e v a s o t o c i n as a r e -s u l t of one mutation. The formation of 8-valine o x y t o c i n would have r e q u i r e d an int e r m e d i a t e compound such as 8-glycine o x y t o c i n or o x y t o c i n i t s e l f ( 8 - l e u c i n e o x y t o c i n ) . On the other hand o x y t o c i n i s al s o f a i r l y widespread. It has been i d e n t i f i e d i n mammals and b i r d s and there are r e -port s of i t s p o s s i b l e occurrence i n r e p t i l e s , amphibians, l u n g f i s h , and i n the h o l o c e p h a l i a n Hydrolagus c o l l e i . I f i t i s assumed t h a t the presence of the same peptide i n two species indicates, i t s presence i n a common ancestor then i t fo l l o w s that o x y t o c i n must have appeared before the dichotomy between the l i n e l e a d i n g to the c a r t i l a g i n o u s f i s h on one hand and that l e a d i n g to a l l other jawed v e r t e b r a t e s on the other (Sawyer, 1968). I f o x y t o c i n i s indeed the p r i m i t i v e molecule, both 8 - v a l i n e and 8-serine o x y t o c i n could have a r i s e n from i t by a s i n g l e mutation. I f one of the peptides should be 8-valine o x y t o c i n , then the s e r i n e residue would be found i n the second p e p t i d e . 198 Apart from g l u t amine•the only amino a c i d i n the k p o s i t i o n of the n a t u r a l l y o c c u r r i n g neurohypophysial peptides i s i n f a c t s e r i n e , which i s found i n i s o t o c i n of the t e l e o s t s and i n g l u m i t o c i n of some elasmohranchs. The second peptide might then be expected to be a ^ - s e r i n e analogue. Should the f i r s t peptide prove to be 8-valine o x y t o c i n and the second to have a U-serine r i n g , then there are two p o s s i b i l i t i e s f o r the s t r u c t u r e of the second p e p t i d e . These are 3-leucine, ^ - s e r i n e , 8-alanine o x y t o c i n or e l s e 3-alanine, ^ - s e r i n e , 8-leucine o x y t o c i n . A 3-leucine s u b s t i t u t i o n g r e a t l y reduces a c t i v i t y , and an 8-alanine s u b s t i t u t i o n a l s o has t h i s e f f e c t (Sawyer, 19&5; Berde and Boissonnas, 1966) ; the unknown peptide accompanying 8-valine o x y t o c i n would be expected to have low a c t i v i t y . The e f f e c t of a 3-alanine s u b s t i t u t i o n on neurohypophysial peptide a c t i v i t y i s not known; 8-leucine occurs i n o x y t o c i n i t s e l f and so t h i s s t r u c -ture would not be expected to a f f e c t the a c t i v i t y of the p e p t i d e . 3-alanine, ^ - s e r i n e , 8-leucine o x y t o c i n i s the more co n s e r v a t i v e molecule since both ^ - s e r i n e and 8-leucine s t r u c t u r e s are known to occur n a t u r a l l y . A l t e r n a t i v e l y , i f the f i r s t p eptide should be 8-se r i n e o x y t o c i n then the second peptide would have one of the s i x p o s s i b l e arrangements of l e u c i n e , v a l i n e , and a l a n i n e i n p o s i t i o n s 3, ht and 8. Despite many i n t e r e s t i n g s p e c u l a t i o n s , the true 1 nature of the p r i n c i p l e s must remain unknown u n t i l 199 a f i n a l s e p a r a t i o n of the two p o s s i b l e components i s achieved, probably by column p a r t i t i o n chromatography. Addendum: After the completion of this investigation, Acher et_ a l . (1972) separated two oxytocic principles from Squalus acanthias by chromatoelectrophoresis. This study supported the presence of the mixture of principles found here. Amino acid analysis and partial Edman degradation suggested the following structures for these principles: 8-valine oxytocin ("valitocin") and 4-asparagine oxytocin ("aspartocin"). Ref: Acher, R., J. Chauvet, and M.T. Chauvet. (1972). "Evolution moleculaire - identification de deux nouvelles hormones neurohypophysaires, la Valitocine (Val 8- ocytocine) et 1'Aspartocin (Asn^-ocytocine) chez un poisson selacien, l'Aiguillat (Squalus acanthias)," C.R.Acad.Sc. (Paris), 274:313-316 Chapter 5 SUMMARY The v e r t e b r a t e neurohypophysis g e n e r a l l y contains at l e a s t two p r i n c i p l e s - a b a s i c , a n t i d i u r e t i c peptide and a n e u t r a l , o x y t o c i c agent (see Sawyer, i 9 6 0 ) . The l e v e l s of a c t i v i t y i n the elasmobranch neurointermediate lobe,as d e t e r -mined by the standard bioassay t e c h n i q u e s , are so low (see Perks and Dodd, 1963a; Perks, 1966; t h i s t h e s i s , Tables IV and X) that e a r l y workers had d i f f i c u l t y i n determining the pre s -ence of any neurohypophysial p r i n c i p l e s i n these f i s h ( H e r r i n g , 1908a; 1908b; 1913; 1915; Waring and Landgrebe , 1950). How-ever, at the time t h i s study was begun the ex i s t e n c e of elasmobranch o x y t o c i n - l i k e agents had been f i r m l y e s t a b l i s h e d , although t h e i r exact number and nature was u n c e r t a i n ; a l i t t l e l a t e r , t r a c e s of a b a s i c p r i n c i p l e were observed i n both skate and shark species (Maetz et_ al_. , 1959; Sawyer et_ S L I . , 1 9 6 l ; H e l l e r and P i c k e r i n g , I961 ; Perks and Dodd, 1963a; 1963b; Sawyer, 1965 ; Perks and Sawyer, 1965 ; Acher et_ a l . , 1965 ; Chauvet et_ a l . , 1965). The neurohypophysial p r i n c i p l e s produced by two elasmo-branch species - the skate, Rai a rhina,. and the d o g f i s h shark, Squalus acanthias ( P a c i f i c v a r i e t y ) -• were examined here. 200 201 The neurointermediate lobes of both species were found to con t a i n low l e v e l s of a c t i v i t y , ranging from 12 to 32 mU/mg d r i e d t i s s u e ( r a t uterus a c t i v i t y , no Mg + +) f o r Squalus , and from 1 to 3 mU/mg d r i e d t i s s u e ( r a t uterus a c t i v i t y , no Mg + +) i n the case of Raia.. In both species a small amount of ba s i c peptide could be separated from the major n e u t r a l f r a c t i o n by chromotography on CM Sephadex. The b a s i c p r i n c i p l e of Squalus appeared to be the widespread p e p t i d e , a r g i n i n e v a s o t o c i n . This i d e n t i f i c a -t i o n was based on the chromatographic and pharmacological s i m i l a r i t y of the p r i n c i p l e to s y n t h e t i c a r g i n i n e v a s o t o c i n ; i n p a r t i c u l a r , i t was based on the new technique of d i r e c t p harmacological comparison of the p r i n c i p l e to s y n t h e t i c a r g i n i n e v a s o t o c i n . The b a s i c p r i n c i p l e was i s o l a t e d from Rai a i n only t r a c e amounts, and i t was not s t u d i e d f u r t h e r ; however i t was t e n t a t i v e l y i d e n t i f i e d as a r g i n i n e v a s o t o c i n on the b a s i s of the s i m i l a r i t y of i t s behavior on i o n exchange chromatography to the b a s i c p r i n c i p l e of Squalus. A v a r i e t y of n e u t r a l neurohypophysial p r i n c i p l e s -EOP I, EOP I I , g l u m i t o c i n , the E.^  p r i n c i p l e s , and the p r i n c i p l e s - had been r e p o r t e d among the elasmobranchs. These p r i n c i p l e s have been d i s t i n g u i s h e d on the b a s i s of t h e i r magnesium p o t e n t i a t i o n values (R M ) or t h e i r r e l a t i v e m i l k -e j e c t i o n a c t i v i t i e s (R..„): g l u m i t o c i n and the E p r i n c i p l e ( i n some spec i e s ) have been c h a r a c t e r i z e d by t h e i r high values 2 0 2 f o r R__ , and EOP II has been d i s t i n g u i s h e d from EOP I on the Mg • b a s i s of i t s high R..„ v a l u e . Mii j However, the ob s e r v a t i o n s made here may i n v a l i d a t e these d i s t i n c t i o n s . When a s e r i e s of crude neurointermediate lobe e x t r a c t s of Raia r h i n a were assayed on the r a t u t e r u s , both i n the presence of magnesium ions and i n t h e i r absence, the r e s u l t i n g magnesium p o t e n t i a t i o n values (^ Mg) "were found to cover an unexpectedly wide range. F u r t h e r i n v e s t i g a t i o n showed that the potency of a s i n g l e e x t r a c t could vary s i g -n i f i c a n t l y , whether assayed on the r a t uterus i n the presence or i n the absence of magnesium i o n s ; when p a i r s of e x t r a c t s which had shown widely d i f f e r e n t p o t e n t i a t i o n s were re-assayed j o i n t l y on the same assay p r e p a r a t i o n s , the p o t e n t i a t i o n s of the two e x t r a c t s converged to c l o s e l y s i m i l a r v a l u e s . These r e s u l t s suggested that the v a r i a b i l i t y i n . potency was due to anomalies i n the d i s t i n c t i o n which d i f f e r e n t assay p r e p a r a t i o n s may make between the unknown peptide and.the o x y t o c i n standard. Some v a r i a b i l i t y was a l s o observed i n the m i l k - e j e c t i o n p o t e n c i e s of both Rai a r h i n a e x t r a c t and of s y n t h e t i c glumi-t o c i n , and t h i s was r e f l e c t e d i n the corresponding r a t i o s of m i l k - e j e c t i o n a c t i v i t y to r a t uterus a c t i v i t y (R ). The v a r i a b i l i t y found i n these two r a t i o s , when they are obtained by independent " c l a s s i c a l " methods of assay, suggests t h a t while they can be used to d i s t i n g u i s h neuro-hy p o p h y s i a l peptides from o x y t o c i n i t s e l f , they are an 2 0 3 u n r e l i a b l e c r i t e r i o n f o r comparing two unknowns- at l e a s t i n the case of elasmobranch e x t r a c t s . Nevertheless assay methods could be used i n e s t a b l i s h i n g the probable i d e n t i t y of an unknown by comparing the p r i n c i p l e d i r e c t l y with s y n t h e t i c p e p t i d e s other than o x y t o c i n ; two i d e n t i c a l p e p t i d e s , assayed side by s i d e , would have the same r e l a t i v e a c t i v i t y on every assay p r e p a r a t i o n and on each type of assay. The v a r i a b i l i t y found here f o r the values of R,,_ and ME R„ , together with the other data a v a i l a b l e at t h i s time, Mg r a i s e d the i n t e r e s t i n g p o s s i b i l i t y that EOP I, EOP I I , E^ , and g l u m i t o c i n might prove to be the same, and that the v a r i e t y of elasmobranch p r i n c i p l e s might be reduced to only two - E^ and g l u m i t o c i n . However, i t must be noted that although the l a t e r s t u d i e s on Rai a were c o n s i s t e n t with t h i s p o s s i b i l i t y , the s t u d i e s on Squalus i n d i c a t e d that i t s n e u t r a l f r a c t i o n c o n s i s t e d of a mixture of two p e p t i d e s , n e i t h e r of which has been p r e v i o u s l y demonstrated. There was no evidence f o r the presence of the chroma-t o g r a p h i c a l l y slow-moving agent, E^, i n e i t h e r s p e c i es s t u d i e d here, although t h i s p r i n c i p l e had been r e p o r t e d to occur both i n other species of skate, and i n Squalus ac a n t h i as ( H e l l e r and P i c k e r i n g , l Q 6 l ; H e l l e r and Roy, 1965a). Amino a c i d analyses of the Rai a r h i n a p r i n c i p l e i n d i c a t e d the presence of a s i n g l e p e p t i d e , g l u m i t o c i n . - Paper chromatography of both Squalus crude e x t r a c t and the p u r i f i e d Squalus n e u t r a l 2 0 U f r a c t i o n i n n - t m t a n o l : a c e t i c acid:water = U : 1 : 5 (the solvent system used hy H e l l e r to r e s o l v e from E ) showed a s i n g l e f a s t - r u n n i n g o x y t o c i c peak, with no evidence f o r the presence of the E^ p r i n c i p l e i n i t s t a i l i n g edge. In one case there was a t r a c e of slower-running p r i n c i p l e , hut t h i s agent showed the pharmacological c h a r a c t e r i s t i c s ( i n p a r t i c u l a r , the high r e l a t i v e f r o g bladder a c t i v i t y ) of a r g i n i n e v a s o t o c i n . I t was p o s s i b l e that an E^ f r a c t i o n c ould have been l o s t during the p r o c e s s i n g of the glands or of the e x t r a c t . However, the small amount of a c t i v i t y which was l o s t during these procedures could not account f o r the high p r o p o r t i o n of the t o t a l a c t i v i t y which has been a t t r i b u t e d to the E^ p r i n c i p l e . The r e s u l t s of the study of the n e u t r a l p r i n c i p l e of Raia r h i n a were c o n s i s t e n t with a s i n g l e s t r u c t u r e f o r EOP I and g l u m i t o c i n . On f i r s t p h a r m a c o l o g i c a l examination the agent appeared to be of the EOP I type, which has been d i s -t i n g u i s h e d from the EOP II p r i n c i p l e by i t s lower values f o r R . Further p h a r m a c o l o g i c a l study, i n c l u d i n g d i r e c t com-M E p a r i s o n of the e x t r a c t to the s y n t h e t i c p e p t i d e , p u r i f i c a t i o n s t u d i e s , and amino a c i d a n a l y s e s , s t r o n g l y i n d i c a t e d t h at t h i s p r i n c i p l e was g l u m i t o c i n . G l u m i t o c i n had p r e v i o u s l y been i d e n t i f i e d i n other species of skate. The n e u t r a l f r a c t i o n of the d o g f i s h Scjualus acanthias appeared to d i f f e r from g l u m i t o c i n and from a l l other neuro-hypo p h y s i a l p r i n c i p l e s which have been i d e n t i f i e d i n nature. 205 I n i t i a l l y , p harmacological s t u d i e s suggested that i t resembled the n e u t r a l p r i n c i p l e found i n many species of sharks and i n most skates, and designated as EOP I. However, while the EOP I p r i n c i p l e of c e r t a i n skates proved to he g l u m i t o c i n , d i r e c t comparison between the p u r i f i e d Squalus f r a c t i o n and s y n t h e t i c g l u m i t o c i n showed c l e a r l y that these two p r i n c i p l e s were not i d e n t i c a l . Amino a c i d analyses of the p u r i f i e d n e u t r a l f r a c -t i o n suggested that i t c o n s i s t e d of an equimolar mixture of two p e p t i d e s . I t should be noted t h a t these two peptides would not correspond to a mixture of E^ and ; there was no evidence f o r the presence of the E^ p r i n c i p l e i n the e x t r a c t . A l s o , the two peptides suggested by the amino a c i d analyses do not correspond i n d i v i d u a l l y to the two n e u t r a l peaks which were e l u t e d o f f the CM Sephadex; no d i f f e r e n c e could be shown between the neurohypophysial a c t i v i t i e s of the two CM Sephadex peaks and so they were combined f o r f u r t h e r analyses. Chromatographic data and t h e o r e t i c a l c o n s i d e r a t i o n s suggested t h a t n e i t h e r of the two p o s t u l a t e d Squalus p r i n c i p l e s was g l u m i t o c i n or any other known neurohypophysial p e p t i d e . The high r e l a t i v e f r o g bladder a c t i v i t y of the n e u t r a l f r a c t i o n suggested that one of the two p r i n c i p l e s might possess an i n t a c t o x y t o c i n r i n g , and based on the l i m i t e d data a v a i l a b l e at p r e s e n t , 8 - v a l i n e o x y t o c i n or 8 - s e r i n e o x y t o c i n were suggested as p o s s i b l e s t r u c t u r e s f o r t h i s p e p t i d e . However, u n t i l the two p o s t u l a t e d p r i n c i p l e s can be separated and i n d i v i d u a l l y analyzed, t h e i r t r u e natures must remain unknown, 206 and any i n f e r e n c e s that might be drawn concerning t h e i r s t r u c t u r e must be regarded as s p e c u l a t i o n . The presence of g l u m i t o c i n i n the skates and of a mixture of two new p e p t i d e s i n the d o g f i s h shark r a i s e s s e v e r a l i n t e r e s t i n g p o s s i b i l i t i e s . F i r s t l y , there may be a wide-spread d i f f e r e n c e between the p r i n c i p l e s produced by the skates and the sharks. Secondly, i t i s p o s s i b l e that not only the skates, but a l s o those other species which have a d i f f u s e type of pars nervosa (see M e u r l i n g , 1967a) may sy n t h e s i z e g l u m i t o c i n , while species such as Squalus which have a d i s c r e t e pars nervosa s t r u c t u r e (see M e u r l i n g , 1962) may e l a b o r a t e a mixture of p e p t i d e s . However, s t u d i e s on the neurohypophysial p r i n c i p l e s produced by many more elasmo-branch species i s r e q u i r e d before any such g e n e r a l i z a t i o n s w i l l be p o s s i b l e . BIBLIOGRAPHY Acher, R., and J . Chauvet. (1953). "La s t r u c t u r e de l a va s o p r e s s i n e de boeuf," Biochim. Biophys. A c t a , 12: 1+87-488. Acher, R., J . Chauvet, M. T. L e n c i , F. Morel, and J . Maetz. ( i 9 6 0 ) . 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The d i f f e r e n t i a l equation of s o l u t e c o n c e n t r a t i o n s at a steady s t a t e and i t s s o l u t i o n f o r simple cases," Acta Chem. Scand. , 15:325-31+1.. 218 Svensson, H. (1962a). " i s o e l e c t r i c f r a c t i o n a t i o n , a n a l y s i s , and c h a r a c t e r i z a t i o n - of ampholytes i n n a t u r a l pH g r a d i e n t s . 11. B u f f e r i n g c a p a c i t y and conductance of i s o i o n i c ampholytes," Acta Chem. Scand. , 16 : i)- 5 6 — 1+66. Svensson, H. (1962b). " i s o e l e c t r i c f r a c t i o n a t i o n , a n a l y s i s , and c h a r a c t e r i z a t i o n of ampholytes i n n a t u r a l pH g r a d i e n t s . 111. D e s c r i p t i o n of apparatus f o r e l e c t r o -l y s i s i n columns s t a b i l i z e d by d e n s i t y g r a d i e n t s and d i r e c t d etermination of i s o e l e c t r i c p o i n t s , " Arch. Biochem. Biophys., Suppl. 1:132-138. Swi a t k i e w i c z , V. J . (1967). "Studies of the o x y t o c i c a c t i v i t y of- the neurointermediate lobe of Squalus acanthias ( P a c i f i c v a r i e t y ) " (M.Sc. t h e s i s , U n i v e r s i t y of B r i t i s h Columbia). Van Dyke, H. B., and A. B. Hastings. (1928). "The response of smooth muscle i n d i f f e r e n t i o n i c environments," Am. J . P h y s i o l . , 8 3 : 5 6 3 - 5 7 7 . Van Dyke, H. B., B.'F. Chow, R. 0. Greep, and A. Rothen. (19I+2) "The i s o l a t i o n of a p r o t e i n from the pars n e u r a l i s of the ox p i t u i t a r y with constant o x y t o c i c , p r e s s o r and" d i u r e s i s - i n h i b i t i n g a c t i v i t i e s , " J . Pharmacol. E x p t l .  Therap . , 7l+:190-209. Van Dyke, H.B., K. Adamsons J r . , and S. L. Engel. (1955). "Aspects of the bi o c h e m i s t r y and p h y s i o l o g y of the neurohypophysial hormones," Recent Progress i n Hormone  Re search , 11:1-1+1. Ve s t e r b e r g , 0., and H. Svensson. (1966). " i s o e l e c t r i c f r a c t i o n a t i o n , a n a l y s i s , and c h a r a c t e r i z a t i o n of ampholytes i n n a t u r a l pH g r a d i e n t s . IV. Further s t u d i e s on the r e s o l v i n g power i n connection with s e p a r a t i o n of myoglobins," Acta Chem. Scand., 20:820-834. V i z s o l y i , E., and A. M. Perks. (1969). "New neurohypophysial p r i n c i p l e i n f o e t a l mammals," Nature, 223:1169-1171. V l i e g e n t h a r t , J . F. G., and D. H. G. Versteeg. (1967). "The e v o l u t i o n of the v e r t e b r a t e neurohypophysial hormones i n r e l a t i o n to the genetic code," J . E n d o c r i n o l . , 38:3-12. Vogt, M. (1953). "Vasopressor, a n t i d i u r e t i c , and o x y t o c i c a c t i v i t i e s of e x t r a c t s of the dog's hypothalamus," B r i t . J . Pharmacol. , 8:193-196. 219 Von den Velden, R. (1913). "Die Nierenwirkung von Hypophysenextrakten bein Menschen," K l i n . Wschr., 50: 2083.' C i t e d by H. H e l l e r . (1963). "Pharmacology and d i s t r i b u t i o n of neurohypophysial hormones," Symp. Zoo l . Soc. London, 9:93-106. Walter, R., B. Dubois, and I. Schwartz. (1968). " B i o l o g i c a l s i g n i f i c a n c e of the amino a c i d r e s i d u e i n p o s i t i o n 3 of neurohypophysial hormones and the e f f e c t of mag-nesium on t h e i r u t e r o t o n i c a c t i o n , " E n d o c r i n o l o g y , 83:979-983. Waring, H., and F. W. Landgrebe. (1950). "Hormones of the p o s t e r i o r p i t u i t a r y , " The Hormones (G. Pincus and K. V. Thimann, eds.). New York: Academic P r e s s . V o l . 2, pp. 427-514. Wilson, N. (1968). " I s o l a t i o n and amino a c i d sequence of neurohypophysial hormones of P a c i f i c chinook salmon (Oncorhynchus tschawytscha)" (Ph.D. t h e s i s , U n i v e r s i t y of B r i t i s h Columbia). Wilson, N., and M. Smith. (1969). " i s o l a t i o n and amino a c i d sequence of neurohypophysial hormones of P a c i f i c chinook salmon (Oncorhynchus tschawytscha), " Gen. Comp . E n d o c r i n o l . , 13 :1+12-1+24 . A P P E N D I X VI Source of the Neurointermediate Glands used i n t h i s Study Sample Loc a t i o n of catch Date Storage Number T o t a l Ave rage number of i n of weight weight Cat ch dry i c e glands of per (DI) or glands gland acetone(A) (mg) (mg) (the skate, Raia rhina) 1 Kyugout Channel Aug. '66 A 11 132.9 12. 1 2 Oukounish Channel Aug. '66 A 18 211. 9 l l . 8 3 West coast of Vancouver I s l a n d Aug. '66 A 1+ 1+9.3 12 . 3 1+ Nanaimo, B.C. J u l y '66 A 3 39.0 13. 0 5 La Perouse Banks Aug. •66 A 33 1+36 .1 13. 2 6 La Perouse Banks Aug . •66 A 15 193.1+ 12. 9 7 South of Leonard Islands Aug. •66 A 13 235 .6 18. 1 8 La Perouse Banks Aug. •66 A 18 206.6 11. 5 9 La Perouse Banks Aug . •66 A 33 370.9 11. 2 10 30 miles o f f Cape Scott Aug. '66 A 1+ 51.6 12. 9 11 30 miles o f f Cape Scott Aug. •66 A 10 78.2 7. 8 12 Kyugout Channel Aug. •66 A 6 70. 8 11. 8 13 Stuart Channel Jan. '67 A 18 14 Pylades Channel Jan . •67 A 30 15 Point Grey, Vancouver Feb . '67 A 1 16 Nanaimo, B.C. Feb . '67 A 3 IT Nanaimo, B.C. Mar. '67 A 2 18 Dod 1s Narrows Mar. •67 A 5 2166.6 8. 6 19 La Perouse Banks Mar . •67 A 55 20 Dod 1s Narrows June •67 A 10 21 F r i d a y Harbor, U.S.A. Apr. •68 DI 13 22 F r i d a y Harbor, U.S.A. May •68 DI 111 23 Steveston, B.C. Aug. '68 DI 3 24 Steveston, B.C. Oct. '68 DI 1 APPENDIX--1 continued Sample Lo c a t i o n of catch Date Storage Numbe r T o t a l Average number of i n of weight we i ght Catch dry i c e glands of per (DI) or glands gland acetone(A) (mg) (mg) (the d o g f i s h , Squalus acanthias i ( P a c i f i c v a r i e t y ) 25 Nanaimo, B.C. J u l y '66 A 21 181.6 8.7 26 Nanaimo, B.C. Aug. '66 A 26 232.5 8.9 27 West coast of Vancouver I s l a n d Aug . '66 A 12 68.0 5.7 28 La Perouse Banks Aug. '66 A 54 333.6 6.2 29 Roberts Banks Nov. '66 A 50 245.0 4.9 30 Ladysmith , B.C. Jan . '67 A b9 326. 4 6.7 31 Nanaimo, B.C. Mar. •67 A 32 291.2 9.1 32 Nanaimo, B.C. Mar. •67 A 91 718.9 7.9 33 La Perouse Banks Mar. '67 A 299 1722.8 5.8 34 La Perouse Banks Mar. '67 A 219 1321.1 6.0 35 E v e r e t t , U.S.A. Mar. •67 A 50 340. 8 6.8 36 E v e r e t t , U.S.A. Mar . '67 A 79 613.2 7.8 37 E v e r e t t , U.S.A. Mar . •67 A 20 171. 3 8.6 38' E v e r e t t , U.S.A. Mar . '67 A 53 433. 0 8.2 St andards APPENDIX II used i n B i o l o g i c a l Assays 1. S y n t h e t i c o x y t o c i n (Syntocinon, Sandoz Pharmaceuticals), 10 IU/ml. D i l u t e d to 5"<mU/ml and 10 mU/ml f o r the r a t uterus assay (both i n the' presence and i n the absence of Mg ); d i l u t e d to 100 mU/ml f o r the r a b b i t m i l k - e j e c t i o n assay; used u n d i l u t e d i n volumes of 25 and 50 .pi f o r the f r o g bladder assay. 2. Synthet i c g l u m i t o c i n ( s u p p l i e d by Dr. M. Manning, M c G i l l U n i v e r s i t y through the kindness of Dr. ¥. Sawyer, Columbia U n i v e r s i t y ) , d i s s o l v e d at 1 mg/ml i n an aqueous 0.25$ s o l u -t i o n of a c e t i c a c i d c o n t a i n i n g 0.5% c h l o r o b u t a n o l . D i l u t e d to 1 ,]!ig/ml f o r the r a t uterus assay; d i l u t e d to l.H y.g/ml f o r the r a b b i t m i l k - e j e c t i o n assay; d i l u t e d to 0.01 mg/ml f o r the r a t a n t i d i u r e t i c assay. 3. S y n t h e t i c a r g i n i n e v a s o t o c i n ( k i n d l y s u p p l i e d by Dr. B. • Berde , Sandoz L a b o r a t o r i e s ) , 0.1 mg/ml. D i l u t e d to 0.1 yg/ml f o r the r a t uterus assay and f o r the f r o g bladder assay; d i l u t e d to 0.0025 ipg/ml f o r the r a t a n t i d i u r e t i c assay. k. A r g i n i n e / l y s i n e v a s o p r e s s i n ( P i t r e s s i n , Parke D a v i s ) , 20 IU/ml. D i l u t e d to 0 ..5 mU/ml f o r the' r a t a n t i d i u r e t i c as s ay. 222 S o l u t i o n s APPENDIX I I I used i n B i o l o g i c a l Assays 1. Rat Uterus Assay: Bathing S o l u t i o n The bathing s o l u t i o n was formulated by van Dyke and Hastings (1928) and mod i f i e d by Munsick ( i 9 6 0 ) . The follow-ing aqueous stock s o l u t i o n s were prepared: a. van Dyke-Hastings s o l u t i o n NaCl 120.67 gm NaHC0 3 1+6.62 gm KC1 8.275gm 0.02$ Na p h e n o l s u l f o n e p h t h a l e i n 270 ml made to 18 l i t e r s b. phosphate b u f f e r , pH 7.1+ i ) 22.1lh gm Na 2HP0^ , made to 1 l i t e r , i i ) 6.31+9 gm NaHgPO^. H 20, made to 1 l i t e r . S o l u t i o n ( i i ) was added to s o l u t i o n ( i ) u n t i l the pH of the combined s o l u t i o n reached 1.k (Radiometer, pH meter type PHM 22). c. 0.5 M C a C l 2 and 0.5 M MgClg For each s a l t an approximately 1 M s o l u t i o n was prepared and i t s m o l a r i t y was determined p r e c i s e l y by measurement of i t s c h l o r i d e c o n c e n t r a t i o n . This was done with a c h l o r i d i m e t e r (Aminco Cotlove C h l o r i d e T i t r a t o r , American Instrument) or by manual t i t r a t i o n against an AgNO, 2 2 3 22k APPENDIX I I I (continued) s o l u t i o n (Fajans t i t r a t i o n ) as o u t l i n e d "by Hoar and Hickman (1967). The s o l u t i o n was then d i l u t e d a p p r o p r i a t e l y to a 0.5 M c o n c e n t r a t i o n . The above stock resolutions were mixed to form the f i n a l b a t h i n g s o l u t i o n , with or without the presence of magnesium i o n s , a c cording to the f o l l o w i n g f o r m u l i : -M g + + present Mg + + absent van Dyke-Hastings s o l n . phosphate b u f f e r 0.5 M CaCl 0. 5 M MgClgV glucose 1976 ml 20 ml 2 ml 2 ml 1 gm 1978 ml 20 ml 2 ml 1 gm The s o l u t i o n s were gassed with a mixture of 5% CO^ i n a i r or oxygen u n t i l the pH reached 7•k. This was deter-mined v i s u a l l y from the col o u r of the i n d i c a t o r present i n the s o l u t i o n . 2. M i l k - E j e c t i o n Assay: D i a l A n a e s t h e t i c The a n a e s t h e t i c , prepared a c c o r d i n g to d i r e c t i o n s from the Ciba Co. (personal communication to Dr. A. M. P e r k s ) , c o n s i s t e d of monoethyl urea (10 gm), e t h y l urethane (10 gm), and d i a l l y l b a r b i t u r i c a c i d (2.5 gm) d i s s o l v e d i n water to a t o t a l volume of 25 ml over a warm (50°C) water bath. The s o l u t i o n was f i l t e r e d through Whatman #1 paper and st o r e d i n 225 APPENDIX I I I (continued) the c o l d (1+°C). 3. Frog Bladder Assay: Ringer's S o l u t i o n The Ringer's s o l u t i o n was that used hy Sawyer ( i 9 6 0 ) . Two aqueous stock s o l u t i o n s were prepared: Stock A NaCl KC1 CaCl .2H 0 9h • 3.7 5.3 gm gm gm made to 1 l i t e r . Stock B NaHC0 3 glue o s e 1+0 gm NaHgPO^.H20 1+ gm 1.77 gm phenol red 12 made to 1 l i t e r . For each assay, 50 ml of stock A and 50 ml of stock B were mixed and made to 1 l i t e r with water. S o l u t i o n s Used In APPENDIX IV N o n - B i o l o g i c a l Measurement s 1. Lowry Peptide Determination a. A l k a l i n e copper s o l u t i o n . 1 ml of 0.5% CuSO^^HgO i n 1% potassium t a r t r a t e was mixed with 5 0 ml of 2% N a 2 C 0 3 i n 0 . 1 N NaOH. h. Phosphomolybdic-phosphotungstic a c i d reagent. 2 N F o l i n C i o c a l t e u Phenol reagent ( F i s h e r Laboratory Chemical) was d i l u t e d 1 :1 with water. 2 . I s o e l e c t r i c Focusing a. Cathode s o l u t i o n . O.k ml of 1ethanolamine was mixed with lk ml water and 1 2 gm sucrose was added. b. Anode s o l u t i o n . 0 . 1 ml s u l f u r i c a c i d was mixed with 1 0 ml water. c. Ampholyte-sucrose f r a c t i o n s . "Ampholine" c a r r i e r ampholytes i n 8% s o l u t i o n , and with pH ranges of 3 - 1 0 or 7 - 9 (LKB Produkter) were used. For each pH range two s o l u t i o n s were prepared: i . Dense s o l u t i o n . 9•k ml of the ampholyte mixture was made to k2 ml with water and 2 8 gm sucrose was added. 2 2 6 APPENDIX IV (continued) 227 i i . Less dense s o l u t i o n . 3.1 ml of the ampholyte mixture was made to 60 ml with water. q The two s o l u t i o n s were combined i n 24 d i f f e r e n t f r a c t i o n s , i n which the volume of the dense s o l u t i o n per f r a c t i o n i n c r e a s e d s t e a d i l y from zero to 4.6 ml. In each case the volume of l e s s dense s o l u t i o n necessary to b r i n g each f r a c t i o n to a f i n a l volume of 4.6 ml was added. The peptide sample to be examined was i n c o r p o r a t e d i n t o one of the middle f r a c t i o n s , and r e p l a c e d i t s own volume of the l e s s dense s o l u t i o n . 

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