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Properties of the myosin-like proteins of sea urchin eggs

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Properties of the myosin-like proteins of sea urchin eggs

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Content PROPERTIES OF THE MYOSIN-LIKE PROTEINS OF SEA URCHIN EGGS A T h e s is P r e s e n t e d to th e D epartm ent o f B io c h e m is try and N u t r i t i o n S chool o f M edicine The U n i v e r s i t y of S o u th e r n C a l i f o r n i a I n P a r t i a l F u l f i l l m e n t o f th e R eq u irem en ts f o r th e Degree M aster o f S c ie n c e fcy W illia m Matthew Connors June 1947 UMI Number: EP41281 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. UMI EP41281 Published by ProQuest LLC (2014). Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 This thesis, written by W illiam M atthew __C oM ors under the guidance of h%$.... Faculty Committee, and a p p ro ved by all its members, has been presented to and accepted by the Council on Graduate Study and Research in partial fulfill ment of the requirements for the degree of MASTER OF SCIENCE Secretary D ate June 1947 Faculty Committee Chairman TABLE OF C O MEETS FOREW ORD ................................................................................................. F r o n tp ie c e CHAPTER PAGE I . INTRODUCTION........................................................................ 1 N ature o f th e p r o b l e m ......................................... 4 S i m i l a r i t y betw een m uscle c o n t r a c t i o n and f e r t i l i z a t i o n .............................. 9 I I . EXPERIMENTAL........................................................................ 10 C o l l e c t i o n and tr e a tm e n t o f th e egg s • • 10 Chem ical c o m p o s itio n o f th e eggs . . . . 11 p r e p a r a t i o n o f the M irsky p r o t e i n . . . 11 D e s c r i p t i o n o f a d i a l y s i s a p p a r a tu s . . 13 P r e p a r a t i o n o f th e Weber s o l u t i o n e x t r a c t s . . . . . ............................................... 15 E f f i c i e n c y o f th e e x t r a c t i o n s • • • • . 16 D e s c r i p t i o n o f u l t r a v i o l e t a b s o r p t i o n t e s t s . . . . . .......................... 19 C o r r e l a t i o n o f a b s o r p t i o n and c h e m ic a l a n a l y s i s d a ta .................................. 20 A b s o r p tio n d a ta o f V ies and Gex . . . . 22 ATP-ase A c t i v i t y ......................................................... 23 Whole egg hom ogenates . .......................... 29 F e r t i l i z e d and u n f e r t i l i z e d e x t r a c t s • 33 The M irsky P r o t e i n .............................................. 35 i i i CHAPTER PAGE M y o s i n ....................................................................................... 35 Summarized r e s u l t s ......................................................... 39 B -G ly c e ro p h o sp h a ta se A c t i v i t y .......................... 39 E l e c t r o p h o r e t i c a n a l y s i s • .......................................... 41 Double R e f r a c t i on of Flow . . . . . . . . 48 B a sic p r i n c i p l e s ....................................................... 48 R e s u lt of t e s t ......................................................... 47 E l e c t r o n M ic ro g ra p h S t u d i e s ................................ 48 I I I . CONCLUSIOHS A ED D1SCUS3I0E......................... 58 IV. SUMMARY......................................... ..... . . .......................... 61 BIBLIOGRAPHY ..................................................................................................... 62 LIST OF TABLES TABLE PAGE I . T o t a l N itro g e n e x t r a c t e d By th e M irsky Method . 18 I I # T o ta l N itr o g e n e x t r a c t e d by th e homogenate t e c h n i q u e . • . 18 I I I . Chem ical a n a l y s i s o f th e eg g p r o t e i n s . . . . 21 IV. Log^Q V alues f o r t h e M irsky P r o t e i n and p r o t e i n s o f V ies and Gex a t v a r i o u s u l t r a v i o l e t wave l e n g t h s ................................................... £6 V. H y d r o ly s is o f ATP by th e f e r t i l i z e d and u n f e r t i l i z e d e g g .....................* ........................................ 31 VI. The e f f e c t o f c a lc iu m and magnesium io n on th e ATP-ase a c t i v i t y o f th e f e r t i l i z e d and u n f e r t i l i z e d egg p r o t e i n e x t r a c t s . . . 34 LIST OF FIGURES FIGURE PAGE 1. U l t r a v i o l e t A b s o r p tio n Spectrum o f th e M ifsky P r o t e i n ............................................... 24 2. U l t r a V i o l e t A b s o rp tio n S p e c tr a o f th e eg g p r o t e i n s .............................................. 25 3* ATP-ASE A c t i v i t y o f Sea U rc h in egg H o m o g e n a te s .............................................................. 32 4 . ATP-ASE A c t i v i t y o f U n f e r t i l i z e d egg P r o t e i n s ........................................................................ 36 5 . ATP-ASE A c t i v i t y of Rat M y o s i n ................................ 38 LIST OF PLATES PLATE PAGE 1. P h o to g ra p h o f t h e D i a l y s i s A p p aratu s . . . . 14 I I . E l e c t r o p h o r e t i c A scendin g Boundary of th e - u n f e r t i l i z e d e g g p r o t e i n s ............................... 43 I I I . E l e c t r o p h o r e t i c A scen ding Boundary of th e u n f e r t i l i z e d egg M irsky P r o t e i n . . . . 44 IV. P h o to g ra p h of th e Assembled Double R e f r a c t i o n o f flo w a p p a r a t u s .................................... 49 V. View of th e A ssem bled C y lin d e r s a n d p o l a r o i d s a p p ro x im a te ly o n e - h a l f a c t u a l s i z e ................................................................................ 50 VI. "E xploded" view of th e C y lin d e r s and O p t i c a l A ttac h m e n ts ......................................................... 51 V II. P h o to g ra p h o f th e M irsky p r o t e i n f i e l d b e f o r e a g i t a t i o n ................................... 52 V I I I . P h o to g ra p h o f th e M irsky p r o t e i n F i e l d d u r in g a g i t a t i o n ...................................................................... 53 IX. E l e c t r o n M ic ro g ra p h of R a b b it M yosin, 75,000 X i n 0 .6 M. KC1, pH 7 . 4 ............................... 56 X. E l e c t r o n M ic ro g ra p h of th e M irsky p r o t e i n from u n f e r t i l i z e d e g g s , 7 5 ,0 0 X .. 0 .1 5 7 p e r c e n t p r o t e i n S o l u t i o n i n 0 .5 M. KOI, pH 7 . 0 ................................................................................................. 57 FOREW ORD I n an e n d e a v o r to s o lv e th e m y s t e r i e s o f v e r t e b r a t e p h y s io lo g y , r e s e a r c h e r s down th ro u g h t h e y e a r s have o f t e n t u r n e d to a stu d y o f th e se em in g ly sim p le u n i c e l l u l a r o rg a n is m s . By su c h an a p p ro a c h i t i s hoped t o g a i n a b a s i c knowledge of th e l i f e p r o c e s s e s i n t h e s e p rim a ry form s ----- and to a p p ly t h i s in f o r m a tio n when d e a l in g w ith th e more com plex. However, one m ust p ro c e e d w ith c a u t i o n when f o l l o w in g su ch a p a t h , as many "sim p le c e l l s " may, in d e e d , c o n t a i n most o f th e sy s te m s o f h i g h e r o rg a n is m s . The s p e c i a l i z e d c e l l s o f th e v e r t e b r a t e s do not alw ays a f f o r d p r o f i t a b l e m a t e r i a l f o r r e s e a r c h i n C e l l u l a r p h y s io lo g y . As a c o n s e q u e n c e , a s h i f t has b e en d i r e c t e d t o e x p e r i m e n ta tio n w i t h th e germ c e l l s of s p e c i e s i n the lo w er p h y l a . H ere, one may w itn e s s s i m p l i c i t y , c o m p le x ity , and e v o l u t i o n ------ a l l i n a s h o r t p e r i o d of tim e . The se a u r c h i n i s su c h a s p e c i e s and i t s 1 eggs a re th e e x p e r im e n ta l m a t e r i a l s . B i o l o g i s t s , B io c h e m is ts , and p h y s i o l o g i s t s , a l i k e , have u se d t h i s t i n y l i f e form as a t o o l i n t h e i r r e s e a r c h and t h e l i t e r a t u r e a t t e s t s to th e s u c c e s s o f t h e i r e f f o r t s . I n th e f o l lo w in g w ork, th e s e a u r c h i n egg was u t i l i z e d to d e m o n s tra te p h y s i c a l phenomena and an ftnzymatic r e a c t i o n common t o th e egg and mammalian m uscle. CHAPTER I PROPERTIES OF THE MYOSIN-LIKE PROTEINS OF SEA URCHIN EGGS INTRODUCTION I n 1936, a . E. M irsky i s o l a t e d u n d e n a t u r e ! p r o t e i n s from th e eggs o f t h e s e a u r c h i n s , S t r o n g y l o c e n t r o t u s p u rp u - r a t u s and A r b a c ia p u n c t u l a t a . I t was p o s s i b l e to b r i n g i n t o s o l u t i o n some 83% o f th e t o t a l p r o t e i n from u n f e r t i l i z e d e g g s , b u t only 70% w ent i n t o s o l u t i o n i f th e eggs were f e r t i l i z e d . Thus, around 13% o f t h e s o l u b l e egg p r o t e i n was r e n d e r e d ni n s o l u b l e n by th e f e r t i l i z a t i o n p r o c e s s . I t was n o te d t h a t t h i s phenomena o c c u r r e d i n a 3 to 10 m inute i n t e r v a l a f t e r f e r t i l i z a t i o n , and t h a t no f u r t h e r change c o u ld be d e t e c t e d w i t h i n th e n e x t £ h o u rs when the f i r s t c le a v a g e ta k e s p la c e * Accompanying th e s o l u b i l i t y d e c r e a s e was an i n c r e a s e i n th e e l a s t i c s t r e n g t h o f the egg. T his was d e m o n s tra te d by a f r e e z i n g and th a w in g t e c h n i q u e . U n f e r t i l i z e d eggs were b ro k en by t h i s p r o c e s s b u t f e r t i l i z e d eggs rem ain ed whole . The d e c r e a s e i n p r o t e i n s o l u b i l i t y was not a s s o c i a t e d w i t h th e e l e v a t i o n of th e f e r t i l i z a t i o n membrane n o r w i t h th e p r o c e s s o f c e l l d i v i s i o n , so M irsky c o n c lu d e d t h a t , a f t e r f e r t i l i z a t i o n , a s k e l e t o n framework was p ro d u ce d i n th e egg 2 c y to p la sm c a p a b le of s u p p o r t i n g m e ta b o lic developm ent* He a ls o r e l a t e d th e change i n p r o t e i n s o l u b i l i t y to t h a t i n duced i n m yo sin , th e m uscle p r o t e i n , by r i g o r . E m phasis was l a i d t o th e " s k e l e t o n fram ew ork” th e o r y by th e work o f Beams and K ing (1936) on A s e a r i s e g g s . I t was found t h a t f e r t i l i z e d eggs r e t a i n e d t h e i r a b i l i t y to c le a v e when c e n t r i f u g e d a t 100,000 X g r a v i t y . I n f a c t , t h i s c le a v a g e even p ro c e e d e d i n some c a s e s d u r in g th e c e n t r i f u g a t i o n . The f o r c e in v o lv e d h e re was s t r o n g enough to s e d i ment s m a ll c o r p u s c u l a r p r o t e i n s . These f a c t s would l e a d one to s p e c u l a t e t h a t th e p r o to p la s m of th e f e r t i l i z e d egg may n o t behave a s a f r e e d i s p e r s i o n o f p r o t e i n p a r t i c l e s . As f u r t h e r e v id e n c e f o r th e th e o r y of s t r u c t u r a l o r i e n t a t i o n d u r in g f e r t i l i z a t i o n , we may r e f e r t o th e work o f Moore and M i l l e r (1937) who i n v e s t i g a t e d th e b i r e f r i n g e n c e o f th e S t r o n g y l o c e n t r o t u s e g g . u n f e r t i l i z e d e g g s were p la c e d u n d e r c r o s s e d H ic o l p rism s a f t e r a p r e l i m i n a iy c e n t r i f u g a t i o n to move th e c y to p la s m ic g r a n u le s to one s id e f o r c l e a r e r v i s i o n . The h y a li n e c y to p la sm a p p e a re d i s o t r o p i c , b u t upon f e r t i l i z a t i o n became b i r e f r i n g e n t w i t h i n 3 m in u te s tim e . The t h e o r i e s and e x p e r im e n ta l e v id e n c e r e g a r d i n g b i r e f r i n g e n c e a n d t h e phenomena of do u b le r e f r a c t i o n o f flow (DRF) i n c e r t a i n p r o t e i n s o l u t i o n s have been d i s c u s s e d a t l e n g t h by E d s a l l (1942) and Mehl (1 9 3 8 ). B r i e f l y , we may 3 s t a t e t h a t i f an o b j e c t a p p e a rs b r i g h t betw een crossed. K ie o ls - i t i s c l e a r t h a t o r i e n t e d p a r t i c l e s are p r e s e n t . I n h i s o r i g i n a l a r t i c l e (1936a) M irsky s t a t e d t h a t i t was a l s o p o s s i b l e to i s o l a t e th e p r o t e i n , t h a t u n d e rg o e s c h an g es i n s o l u b i l i t y , from u n f e r t i l i z e d e g g s. The e x a c t p ro c e d u r e u s e d w i l l be d e a l t w i t h i n t h e n e x t s e c t i o n . The l a b i l e p r o t e i n e x h i b i t e d s tr e a m in g d o u b le r e f r a c t i o n o f flo w ; i t s p a r t i c l e s a p p e a re d h ig h ly e lo n g a te d ; and a 1 .4 $ s o l u t i o n had a v i s c o s i t y 9 .6 tim e s t h a t of w a t e r . The r a t e o f s h e a r i n f l u e n c e d t h i s l a t t e r v a l u e . The i n t e r e s t i n g f a c t a b o u t t h e s e o b s e r v a t i o n s i s t h a t m yosin i s l i k e th e s e a u r c h i n p r o t e i n i n many r e s p e c t s . I t t o o , e x h i b i t s s tr e a m in g d o u b le r e f r a c t i o n o f flow and s t r u c t u r a l v i s c o s i t y . The work o f E d s a l l and Me h i (1940) and M irsky (1938) has shown t h a t i t can be b ro u g h t i n t o a t h i x o t r o p i c s t a t e and w i l l ex h i b i t marked e l a s t i c i t y . The f r a c t i o n i s o l a t e d by M irsky (which w i l l be r e f e r r e d to i n th e f u t u r e a s th e M irsky P r o t e i n ) was o f p rim a ry i n t e r e s t to me i n t h i s s tu d y . I n p a r t i c u l a r , i t was d e c id e d to i n v e s t i g a t e th e m yosin a n a lo g y . W orkers i n th e p a s t have i s o l a t e d " m y o s in - lik e " p r o t e i n s from a v a r i e t y of c e l l s . The " p la s m o s in " o f B en sley and H oerr (1934) and B e n sley (1938) and the " r e n o s in " o f Banga and S z e n t- Gyorgyi (1940) were e x t r a c t e d u n d e r s i m i l a r 4 c o n d i t i o n s a s th e M irsky P r o te in * S tre a m in g d o u b le r e f r a c t i o n o f flo w was l ik e w i s e dem o n strated * However, i n a r e c e n t p u b l i c a t i o n , M irsky and P o l l i s t e r (1946) have once a g a i n b ro u g h t to th e r e a d e r ’ s a t t e n t i o n t h a t th e f i b r o u s m a t e r i a l e x t r a c t e d b y t h e s e w o rk ers owes i t s d o u b le r e f r a c t i o n o f flow (DBF) to th e p re s e n c e of p o ly m e riz e d d e s - o x y r ib o s e n u c l e i c a c i d . Thus th e m yosin a n a lo g y l o o s e s i t s v a lu e i n th e s e c a s e s . MirsJcy d id no t commit h im s e l f on t h e n a t u r e o f h i s s e a u r c h i n p r o t e i n . A s tu d y was u n d e r t a k e n i n t h i s l a b o r a t o r y to more f u l l y c h a r a c t e r i z e th e M irsky P r o t e i n w ith r e s p e c t t o - - 1. A d e n o s in e tr ip h o s p h a ta s e (ATP a se ) a c t i v i t y £ . Chem ical c o m p o s itio n 3. A p p l i c a t i o n o f p h y s i c a l c h e m ic a l t e s t s to d e te rm in e i t s n a t u r e . I n th e t h i r d s t e p i t was p la n n e d to r e p e a t th e DRF t e s t s and a l s o to ru n su c h e x p e rim e n ts a s : u l t r a v i o l e t a b s o r p t i o n sp e c tru m , e l e c t r o p h o r e s i s , and e l e c t r o n m ic ro g ra p h s t u d i e s . I t i s b e li e v e d t h a t no one h as r e p o r t e d a t e s t f o r ATP-ase i n th e s e p r o t e i n s . Much has been s a i d i n th e l i t e r a t u r e r e g a r d i n g t h e i r p h y s i c a l n a t u r e and a l i t t l e work h as been r e p o r t e d a s to th e c h e m ic a l c o m p o s itio n o f " p la s m o s in " and " r e n o s i n " . E n g e lh a r d t and lyubim ova (1939) were th e f i r s t to 5 d e m o n s tra te t h a t m yosin h y d ro ly z e d th e compound ad eno- s i n e t r i p h o s p h a t e (ATP)* l i b e r a t i n g i n o r g a n i c pho sp h ate and a d e n o s in e d ip h o s p h a te (ADP). Most workers, have found m yosin and ATP-ase to be c l o s e l y a s s o c i a t e d . T h e r e f o r e , th e s p e c i f i c i t y f o r ATP s h o u ld be a more t r u e c r i t e r i o n f o r a nm yosin- 1 i k e 1 1 p r o t e i n . DuBois and P o t t e r (1943) d e m o n s tra te d th e p re s e n c e of ATP-ase i n s e v e r a l anim al t i s s u e s b e s i d e s m u sc le . S te in b a c h and Moog (1945 a ,b ) have d e m o n s tra te d ATP-ase i n c h ic k embryos and hen e g g c y to p la s m ic g r a n u l e s . B id d u lp h , Meyer and McShan (1946 a ,b ) have fo u n d th e enzyme i n t h e c o rp o r a l u t e a of th e r a t and have n o te d i t s d e c r e a s e d u r in g a f u n c t i o n a l c y c l e . The f a c t t h a t th e enzyme i s a tim e ATP- ase has b e en n o te d i n a l l c a s e s a s i t was s p e c i f i c f o r th e t e r m in a l p h o sp h a te group o f th e ATP m o le c u le . The term " a p y r a s e ” has been s u g g e s te d f o r an enzyme s p e c i f i c f o r b o th l a b i l e p h o sp h ate g ro u p s. I t has been c la im e d t h a t c e r t a i n p h o s p h a t a s e s , a l k a l i n e i n n a t u r e , a re c a p a b le o f s p l i t t i n g b o th l a b i l e p hosphate g r o u p s . (L ie b k n e c h t (19 3 9 ); Schm idt and T h ann hauser (1943) and A x e lro d (1 9 4 7 ). in . some c a s e s th e s e a u th o r s n o te d t h a t a l l of the p h o sp h a te groups were a t t a c k e d . Mac&eod and Summerson (1946) s t a t e t h a t a c id p h o s p h a ta s e w i l l n o t h y d ro ly z e ATP. I n t h i s c a s e th e enzyme was p a r t i a l l y p u r i f i e d by D r. Gutman. One would be le d to 6 deduce from th e f o r e g o i n g d a t a , t h a t t h e r e a re i n n a tu r e s p e c i f i c A T P-ases, s p e c i f i c A P P -ases, and m o n o e s te r a s e s . These w ould a t t a c k th e ATP m o lecule ( i f c o n ta in e d t o g e t h e r i n a m ix tu r e ) i n the o r d e r o f t h e i r s p e c i f i c i t y . T h e r e f o r e , i n r e p o r t i n g th e h y d r o l y s i s o f s u c h a p h o s p h o r y la te d com pound a s ATP, one s h o u ld he s p e c i f i c i n s t a t i n g th e p e r c e n ta g e o f t o t a l p h o sp h a te rem oved. L in d b e rg (1946) has r e p o r t e d t h a t ATP makes up 31$ o f th e t o t a l p ho sp h ate e s t e r s i n th e s e a u r c h i n egg. W h itley (1946) has i s o l a t e d t h i s compound from th e eg g i n a pure s t a t e . I t i s re a s o n e d t h a t the p re s e n c e o f t h i s com pound m ight p r e c lu d e the e x i s t e n c e o f an A T P-ase. The norm al m e ta b o lic p r o c e s s o f an egg c e l l d u r in g th e r e s t i n g s t a t e w ould n o t r e q u i r e l a r g e amounts o f e n e rg y . However, th e f e r t i l i z e d c e l l w ould r e q u i r e much more energy d u r i n g th e developm ent s t a g e s . I n th e ATP m o lecu le we have a po t e n t i a l so u rc e o f t h a t e nerg y - and i t can be t r a n s f e r r e d by e n zy m atic p r o c e s s e s . P o t t e r and DuBois b e l i e v e t h a t t h i s may be th e c ase i n many, i f n o t a l l , o f th e body c e l l s d u r in g t h e i r more a c t i v e m e ta b o lis m . H e ilb ru n n (1937, 1940) has s t r e s s e d th e r e l a t i o n s h i p o f Ca ++ r e l e a s e d u r in g t h e c o n t r a c t i o n of m u sc le . He t e n d s to f a v o r th e th e o ry t h a t t h e r e i s a d i r e c t a c t i o n of c a lc iu m io n w ith m uscle p ro to p la s m . He s t a t e s t h a t the p ro to p la s m 7 o f m uscle c e l l s i s n o t m arkedly d i f f e r e n t from t h a t of l e s s d i f f e r e n t i a t e d body c e l l s . I t c o n s i s t s of a c o r t e x which i s a g e l and a more or l e s s f l u i d i n t e r i o r . I n t h e c o r t e x we f i n d c a t+ i n a bound s t a t e . C a ^ has been d e m o n s tra te d i n th e c o r t e x o f th e s e a u r c h i n eg g, to o . S c h e e r and S c h e e r (1946) have shown t h a t th e a d d i t i o n o f low o r moder a t e c o n c e n t r a t i o n s o f Ca o f t e n m arkedly i n c r e a s e d t h e r a t e o f a c t i v a t i o n of U r e c h is cauoo ( e c h i u r o i d m arine worm) e g g s. Membrane e l e v a t i o n i s u s u a l l y t a k e n a s a c r i t e r i o n of a c t i v a t i o n . We m ight n o te t h a t a r t i f i c i a l a c t i v a t i o n s do not alw ays r e s u l t i n th e c e l l p r o c e s s e s o f c le a v a g e . H e ilb ru n n has even gone so f a r a s to s u g g e s t t h a t a l l a c t i v a t i o n s i n v o lv e i n t e r n a l l i b e r a t i o n of c a lc iu m i o n s . As f a r a s m uscle c e l l s a re c o n c e rn e d , he t h e o r i z e s , t h a t upon s t i m u l a t i o n Ca++ (c a lc iu m io n ) i s r e l e a s e d , e n t e r i n g t h e c e l l i n t e r i o r and p r o d u c in g a c l o t t i n g o f th e c e l l p r o to p la s m . The ex p e r im e n ta l work o f H e ilb ru n n (1937) and Chambers and Hale (1932) seem to b e a r t h i s o u t. These a u t h o r s d e m o n s tra te d t h a t t h e e n tr a n c e o f C a ++ i n t o th e c e l l i n t e r i o r (m uscle) p ro d u ce d a marked s h o r t e n i n g . I t was n e c e s s a r y to i n j e c t th e io n d i r e c t l y as a d m i n i s t r a t i o n to the u n i n j u r e d e x t e r i o r d id n o t have any r a p i d e f f e c t . H e ilb ru n n (1940) has s t u d i e d th-j e f f e c t of o x a l a t e i o n on se a u r c h i n egg p ro to p la s m , i f one r u p t u r e d th e egg membrane i n a s o l u t i o n of o x a l a t e , the 8 p ro to p la s m m erely m ixed w ith th e s o l u t i o n . However, i f o n ly a s m a ll c u t was made i n t h e egg w h ile i n th e o x a l a t e s o l u t i o n , th e p ro to p la sm would n o t exude from th e c e l l , a s u r f a c e p r e c i p i t a t i o n a c t i o n was v i s u a l i z e d h e r e . I t is s u g g e s te d t h a t o t h e r io n s such a s magnesium le a v e the c e l l more r a p i d l y th r o u g h th e t e a r , h u t th e Caf+ rem a in s i n s i d e , and a t th e t o r n end c a u se s a s u r f a c e p r e c i p i t a t i o n , th u s s e a l i n g o f f th e c e l l . From th e p r o c e e d in g work we can see th e r o l e t h a t c a lc iu m p l a y s i n th e m uscle c e l l and s e a u r c h i n eg g a c c o rd in g to t h e t h e o r i e s o f some a u t h o r s . M irsky (1936 b ) , n o te d a d e c r e a s e i n th e s o l u b l e p o r t i o n o f m yosin, when th e m uscle was i n r i g o r . T his was s i m i l a r to th e change i n s o l u b i l i t y o f s e a u r c h i n egg p r o t e i n s a f t e r f e r t i l i z a t i o n . has been shown t o have an i n h i b i t o i y e f f e c t on th e ATP-ase o f m u sc le , B a ile y (1 9 3 9 ); b u t an a c t i v a t i n g e f f e c t on th e ATPase of th e e l e c t r i c o rg an o f th e e e l , G r e v i l l e and Lehmann (1 9 4 3 ). F o r th e p r e s e n t i t i s b e s t to a g re e w ith th e c o n c l u s i o n o f S c h e e r (1946) t h a t more work i s n eeded a lo n g t h i s l i n e . However, we may form a t e n t a t i v e h y p o t h e s is from w hich to s t a r t . The l i b e r a t i o n o f c a lc iu m io n by an a c t i v a t e d o rg an ism o r s t i m u l a t e d m u scle; th e r e s u l t i n g s t i m u l a t i o n o f an ATPase sy stem ; and th e f o r m a t io n o f an i n s o l u b l e p r o t e i n -• 9 p r o v id e s th e ground w ork. A s e h e ra a tic diagram o f th e s i m i l a r i t y b etw een th e con t r a c t i o n o f m uscle and th e f e r t i l i z a t i o n o f a s e a u r c h i n egg f o l l o w s : MUSCLE CONTRACTION Ca^*- p r o t e i n com plex I e x c i t a t i o n A c t i v a t i o n " I n s o l u b l e ” Myosin SEA URCHIN EGG- FERTILIZATION Ca+ - P r o t e i n P r o t e i n complex U * n - r > » Tl a4* a ■ ! i r \ o A c t i v a t i on ”I n s o l u b l e ” P r o t e i n I t rem a in e d to t e s t f o r th e p o s s i b l e p re s e n c e o f an ATP-ase a d s o rb e d on th e se a u r c h i n " I n s o l u b l e ” P r o t e i n (M irsky P r o t e i n ) o r somewhere w i t h i n th e c e l l i n t e r i o r . CHAPTER I I EXPERIMENTAL ISOLATION METHODS C o l l e c t i o n and, t r e a t m e n t o f th e e g g s . M irsky (1936a) has o u tlin e d , th e g e n e r a l p ro c e d u re u s e d i n h is i s o l a t i o n o f th e p r o t e i n which u n d e rg o e s s o l u b i l i t y changes a f t e r f e r t i l i z a t i o n . He s t a t e s t h a t i t was p o s s i b l e t o o b t a i n i t from b o th f e r t i l e an d u n f e r t i l i z e d e g g s . T h is , I have c o n firm e d . The method u s e d was a s f o llo w s : About 100 se a u r c h i n s (s t r o n g y l o c e n t r o t u s p u r p u r a t u s ) were c o l l e c t e d from th e b r e a k w a te r a t c o ro n a d e l Mar i n May o f 1946. The o v a r i e s were removed and the eg g s added to a b e a k e r o f s e a w a t e r . About 3 /8 o f th e t o t a l amount were t r a n s f e r r e d to a s e p a r a t e v e s s e l where th e y were f e r t i l i z e d w ith sperm . B o th b a tc h e s were t h e n com pacted by c e n t r i f u g i n g and q u ic k ly f r o z e n i n an e t h e r - d r y i c e m ix tu re a t - - 7 7 ° c . The p o r t i o n s were th e n p la c e d in a l y o p h i l e ap p a r a t u s where th e y re m a in e d u n t i l d r y . Such a p ro c e d u re w i l l d i s r u p t th e u n f e r t i l i z e d eg g s but th e f e r t i l e ones re m a in i n t a c t . The p ro d u c ts t h u s o b t a i n e d were th e n gro u n d i n a m o rta r to a f i n e powder. S c a r c e ly an i n t a c t c e l l re m a in e d . The eggs were th e n p la c e d i n g l a s s c o n t a i n e r s , t i g f r t l y c ap p ed , and l e f t i n th e c o ld room u n t i l u s e d f o r th e e x t r a c t i o n . Around £5 grams o f u n f e r t i l i z e d eggs and 15 grams o f f e r t i l i z e d eggs 11 ( n o t c o r r e c t e d f o r m o is tu r e ) were o b ta in e d i n t h i s powdered s t a t e . An a n a l y s i s as t o t h e i r c o m p o s itio n y i e l d e d t h e f o l l o w i n g r e s u l t s ; FERTILIZED EGGS UNFERIILIZED EGGS P e r c e n ta g e P e rc e n ta g e Ash 3 3 .6 3 1 4 .4 3 M o istu re 1 0 .5 4 7 .8 7 N itro g e n 6 .2 5 9 .4 4 P r e p a r a t i o n o f th e M irsky P r o t e i n . T his p r o t e i n was i s o l a t e d from b o th p o r t i o n s o f e g g s a s f o l lo w s ; A w eighed sam ple (3 .8 gram s) o f egg powder was e x t r a c t e d w i t h 500 m l. o f c o ld 1 M KC1, pH 7 . 3 , by g r i n d i n g and s t i r r i n g f o r a b o u t 15 m in u te s i n th e c o ld room. The m a t e r i a l was a llo w e d to s ta n d i n th e c o ld o v e r n i g h t . I n th e m orn in g th e mass was f i l t e r e d th ro u g h a £ in c h l a y e r of nS u p e r -C e ln and t h e n th ro u g h a number 5 Whatman f i l t e r p a p e r, th e pH a t t h i s tim e was s t i l l 7 . 3 . A n o th er f i l t r a t i o n was c a r r i e d out th r o u g h number 5 p a p e r , and th e c l e a r f i l t r a t e t r e a t e d t o a f i n a l c o n c e n t r a t i o n o f £ s a t u r a t i o n w i t h ammonium s u l f a t e . A t t h i s p o i n t i t i s im p o r ta n t t o s t r e s s th e f i n a l c o n c e n t r a t i o n o f ammonium s u l f a t e u s e d . M irsky c la im e d t h a t h i s p r o t e i n came down i n th e f i r s t f r a c t i o n , b u t d i d not s p e c i f y th e c o n c e n t r a t i o n of s a l t u s e d . A p e r s o n a l communi- 12 c a t i o n r e g a r d i n g t h i s p o i n t f a i l e d to c l a r i f y th e i s s u e , i t was th e n u n d e r ta k e n to t r y a s e r i e s o f f r a c t i o n a t i o n s o f t h e e x t r a c t w ith v a r i o u s c o n c e n t r a t i o n s of ammonium s u l f a t e . l / l 6 , l / 8 , l / 4 and l / 2 s a t u r a t i o n s were t r i e d and i t was c l e a r l y e v id e n t t h a t l / 2 s a t u r a t i o n b ro u g h t down the f i r s t p r e c i p i t a t e . I t was a l s o e v id e n t t h a t o t h e r f r a c t i o n a t i o n s o f th e re m a in in g s u p e r n a t a n t by 1/2 s a t u r a t e d s a l t would b r i n g down p r o t e i n s * However, s in c e we were only i n t e r e s t e d i n th e f i r s t f r a c t i o n no f u r t h e r work was done a lo n g t h i s l i n e . The p r e c i p i t a t e d p r o t e i n was a llo w e d to s e t t l e i n t h e c o ld o v e r n i g h t . The pH o f the s o l u t i o n was 5 . 5 . From t h e work o f V ies (1926) we l e a r n t h a t t h e pH of t h e i n t e r i o r o f th e s e a u r c h i n c e l l i s somewhere i n t h e n e ig h b o rh o o d of 5 . 8 . Thus, o u r p r e c i p i t a t i o n may have been n e a r th e i s o e l e c t r i c p o i n t o f th e c e l l p r o t e i n s . The s u p e r n a t a n t f l u i d was d e c a n te d from th e m ix tu re and th e p r o t e i n c e n t r i f u g e d a t room te m p e r a tu re i n i c e d con t a i n e r s . I t was fou nd a d v is a b l e h e re to d i l u t e t h e p r o t e i n m a t e r i a l 1-1 w ith d i s t i l l e d w a te r , o th e r w is e th e h ig h con c e n t r a t i o n o f s a l t w ould keep th e p a r t i c l e s from co m p a ctin g . The com pacted mass was th e n washed 3X w i t h d i s t i l l e d w a te r , su sp e n d e d i n 50 m l. of w a t e r and p l a c e d i n a c o l l o d i a n d i a l y s i s s a c . The m a t e r i a l was d i a l y z e d f o r about 24 hours 13 a g a i n s t c o l d , ru n n in g , d i s t i l l e d w a te r . A s p e c i a l a p a r a t u s was c o n s t r u c t e d dor t h i s s t e p . A p i c t u r e of i t a p p e a rs on th e n e x t page. T his a p p a r a t u s had two s p e c i a l f e a t u r e s : 1 . There was a c o n s t a n t p a ssa g e of c o ld d i s t i l l e d w a te r th r o u g h th e s y s te m , a i d i n g i n rem oving th e l a s t t r a c e s of s a l t . 2. An a g i t a t i n g d e v ic e was s u p p l i e d w hich a l s o a id e d th e p r o c e s s . Around 20 l i t e r s o f d i s t i l l e d w a te r were employed f o r t h e p r o e e s s and th e K e s s l e r t e s t was u s e d as a c r i t e r i o n f o r th e c o m p lete rem oval of ammonium s u l f a t e . The p ro c e d u re f o r o p e r a t i o n was <fuite s im p le . I t was a s f o llo w s ; 1 . P la c e th e d i a l y s i s sa c i n t o th e g l a s s cham ber (3) and th e n f i l l th e v e s s e l a b o u t 3 /4 f u l l w i t h d i s t i l l e d w a te r . 2. A d ju st th e d r i p (2) to a b o u t 1 drop p e r 2 s e c o n d s , and th e n s t a r t th e a g i t a t i n g d e v ic e ( 4 ) . The d i s t i l l e d w a te r was s u p p l i e d from a 5 g a l l o n b o t t l e (n o t shown) by means o f a s ip h o n . A s m a ll m otor s u p p l i e d th e power f o r th e up and down m o tio n o f t h e cham ber. A f t e r t h e p r o t e i n m a t e r i a l had been f r e e d from s a l t i t was made to a c o n c e n t r a t i o n of 0..5 M w ith KCl (pH 7 .0 ) and 100 m l. i n volum e. T e s ts f o r ATP-ase a c t i v i t y (to be d e s c r i b e d l a t e r ) were n e g a tiv e f o r th e p r o d u c t from u n f e r t i l i z e d and f e r t i l i z e d e g g s . A hom ogenate o f th e whole egg m a t e r i a l i n d i c a t e d ATP-ase a c t i v i t y . I t was d e c id e d to e x t r a c t the eggs a t an a l k a l i n e pH, a s i s done f o r m yosin, and th e n t e s t 14 PDASB I PHOTOGRAPH OF THE DIALYSIS APPARATUS 1* D i s t i l l e d w a te r i n t a k e £* D rip o u t l e t 3* G la ss chamber 4* Pow er f o r a g i t a t i n g d e v ic e 15 t h e c o n c e n tr a te d e x t r a c t f o r enzyme a c t i v i t y . I t was r e a s o n e d t h a t i f th e enzyme was a t a l l p r e s e n t i t w ould m a n i f e s t i t s e l f i n th e e x t r a c t . 3 ?he p ro c e d u re u se d i n o b t a i n i n g th e e x t r a c t s (w hich w i l l be r e f e r r e d to i n f u t u r e a s u n f e r t i l i z e d and f e r t i l i z e d ) w i l l be f i r s t g iv e n . F e r t i l i z e d and u n f e r t i l i z e d e x t r a c t s p r e p a r e d w i t h W e b e r n S o l u t i o n . F iv e grams o f eg g powder was homogenized w ith 20 m l. o f a s o l u t i o n known a s W eber’s S o l u t i o n (Weber and Meyer 1 9 3 3 ). The i n s t r u m e n t u s e d was a m o d i f i c a t i o n of P o t t e r ’ s d e s c r i b e d i n nM anom etric T e c h n iq u e s— n (1 9 4 5 ). The e x t r a c t a n t c o n s i s t e d of 0 .6 M KC1 i n m/ 25 HaHCOg and m/ 100 E'agCOg (pH 8 .5 to 8 . 8 ) . A b e a k e r o f c ru s h e d ic e was u s e d to c o o l th e hom ogenizing tu b e d u r in g t h e p r o c e s s . A f t e r th e m a t e r i a l was th o r o u g h ly ground i t was made up to a volume of 50 m l. w i t h th e e x t r a c t a n t . 15 m l. p o r t i o n s from u n f e r t i l i z e d and f e r t i l i z e d eggs w ere s a v e d f o r enzyme t e s t s . The re m a in in g 35 m l ., c o n t a i n i n g 3.5 grams of m a t e r i a l was added to 300 ml. o f t h e Weber e x t r a c t . T h is was s t i r r e d i n the c o ld f o r a b o u t 15 m in u te s and t h e n a llo w e d to s t a n d i n th e c o ld room o v e r n i g h t . I n th e m orning the s u s p e n s io n was f i l t e r e d th r o u g h a l / 4 in c h l a y e r o f " S u p e r-C e l" and th e n tw ic e th ro u g h a Whatman l o . 5 p a p e r . The c l e a r f i l t r a t e was p la c e d i n a v i s c o s e sa c and t r a n s f e r r e d t o the chamber o f th e d i a l y s i s 16 m achine d e s c r i b e d p r e v i o u s l y . The same amounts o f w a te r w ere used a s b e f o r e and th e p e r i o d was f o r 24 h o u r s . The s a c s were removed from th e m achine and th e n c o n c e n tr a te d by e v a p o r a t i n g i n t h e c o ld to a volume o f 75 m l. This was i n t u r n made t o a c o n c e n t r a t i o n of 0 .5 M KC1 (pH 8 .5 ) and 100 ml. i n volum e. The e x t r a c t s a p p e a re d p a le g r e e n i n c o l o r and i t i s b e l i e v e d t h a t th e s u b s ta n c e r e s p o n s i b l e f o r t h i s may have been eeh in o ch ro m e, i t i s p o s s i b l e t h a t i t was r e l e a s e d from th e egg c y to p la s m ic g r a n u le s by th e hom ogenizing t e c h n i q u e . I n th e M irsky e x t r a c t i o n p r o c e d u r e , where only a minimum of g r i n d i n g was c a r r i e d o u t , we d id n o t n o t i c e t h i s g ree n c o l o r . H a r r is (1939) h as s t a t e d t h a t e c h in o e h ro m e , i n th e absen ce o f c e r t a i n s a l t s eg . c a lc iu m and magnesium i n th e medium, w i l l change from r e d to g r e e n . I t i s o f i n t e r e s t to n o te th e p e r c e n t o f t h e t o t a l egg n i t r o g e n removed i n the M irsky p r o t e i n s a n d Weber s o l u t i o n e x t r a c t s . R e fe re n c e i s made to T a b le s I and I I f o r d a ta on th e two m ethods o f e x t r a c t i o n . We s e e t h a t l e s s n i t r o g e n was removed from th e f e r t i l i z e d eg g s i n b o th c a s e s ; a d e c r e a s e o f a p p ro x im a te ly 14 p e r c e n t over th e u n f e r t i l i z e d e g g s , i n t h e l a s t column o f T able I . i t i s n o te d t h a t t h e r e was l e s s n i t r o g e n i n the ammonium s u l f a t e p r e c i p i t a t e o f th e M irsky p r o t e i n . T h is p ro d u c t had been w e l l d i a l y z e d . 17 I n th e l a s t column of Table I I we see t h a t a p p r o x i m a te ly th e same p e r c e n ta g e of th e t o t a l n i t r o g e n was o b t a i n e d f o r b o th t y p e s o f e g g s , a l t h o u g h th e f e r t i l i z e d e x t r a c t con t a i n e d l e s s . T h is d i f f e r e n c e m ight be e x p la in e d on t h e b a s i s o f t h e u n f e r t i l i z e d eggs c o n t a i n i n g more non p r o t e i n n i t r o g e n w hich was d i a l y z e d away. I t may a l s o have b e en t h a t the ho m o g en izin g p ro c e d u re damaged t h e p r o t e i n s . I t i s q u i t e h a rd to r e g u l a t e th e d e g re e of e x t r a c t i o n by M irsky* s m ethod b e c a u se of the g r i n d in g p ro c e d u re em ployed. I t would seem t h a t a more u n ifo rm p r o d u c t would be o b t a i n e d by th e hom o genizing te c h n iq u e . I t was n o t p o s s ib le to f o llo w t h i s p o i n t f u r t h e r b e c a u se o f th e s h o r ta g e of egg m a t e r i a l . i n c o n c l u s i o n , I would s a y t h a t M ir s k y 's s t a t e m e n t r e g a r d i n g the d e c re a s e o f p r o t e i n s o l u b i l i t y i n th e f e r t i l i z e d s e a u r c h i n egg was c o n firm e d when h i s m ethods o f p r e p a r a t i o n w ere u s e d . C hem ical and p h y s i c a l a n a l y s i s o f th e p r o t e i n s . The p o s s i b i l i t y e x i s t e d t h a t th e p r o d u c ts o b ta in e d were n u c le o - p r o t e i n i n n a tu r e so a s e r i e s o f c h e m ic a l a n a l y s i s w ere c a r r i e d o u t. The n i t r o g e n , p h o sp h o ru s , and p e n to se r e l a t i o n s h i p s a r e to be c o n s i d e r e d i n t h i s c a s e . The methods u s e d were t a k e n from "M anom etrie T e c h n iq u e s— n by U m b re it, B u r r i s , TABLE I TOTAL NITROGEN EXTRACTED BY THE MIRSKY METHOD T o ta l N itro g en E x tr a c te d by K .I T o ta l N itro g e n Brought down by Ammonium S u l f a te P r o t e i n M illigram s o f Egg T o ta l N itro g en in e ggs * Per c en t o f Per c e n t of P r e p a r a t io n used M illig ram sP er c e n t M illigram s T o ta l M illigram s T o ta l "Mirsky P r o te in U n f e r t i l i z e d eggs 3,800 358.72 9.44 172.20 48.00 58.60 16.33 "Mirsky p r o t e i n F e r t i l i z e d eggs 3,800 237.50 6 .2 5 80.46 33-38 21.40 9.01 TABLE I I TOTAL NITROGEN EXTRACTED BY THE HOMOGENATE TECHNIQUE T o ta l N itro g en E x tr a c te d by W eber’s S o lu tio n T o ta l N itro g e n in D ia ly z ed E x tr a c t P r o t e in M illigram s o f eggs T o ta l N itro g en in _ 4 _ egg3 ......................................... Per c en t Per c e n t of P r e p a r a tio n used M illigram s Per cent M illig ram s T o ta l M illigram s T o ta l From u n f e r t i l i z e d eggs 3,500 330.40 9.44 221.36 69.66 112.80 34.14 From f e r t i l i z e d eggs 3,500 218.75 6 .2 5 120.32 54.80 75-30 34.42 H oo 19 and S t a u f f e r (1 9 4 5 ). The r e s u l t s a p p e a r i n T able I I I . The r e s u l t s d i d n o t i n d i c a t e any m ajor p o r t i o n o f my p r e p a r a t i o n s to he n u c l e o p r o t e i n . U l t r a v i o l e t a b s o r p t i o n t e s t s were th e n ru n and th e s e r e s u l t s t o g e t h e r w i t h th e c h e m ic a l t e s t s w i l l be c o n s i d e r e d i n t h a t s e c t i o n . U l t r a v i o l e t a b s o r p t i o n s p e c t r a . U l t r a v i o l e t ab s o r p t i o n has been u s e d w id ely a s a q u a l i t a t i v e and q u a n t i t a t i v e a i d i n th e d e t e r m i n a t i o n o f s m a ll am ounts o f su b s t a n c e s i n b i o l o g i c a l f l u i d s . Many o r g a n ic m a t e r i a l s , i n c l u d i n g p r o t e i n s , have c h a r a c t e r i s t i c a b s o r p t i o n bands i n t h i s ra n g e of th e sp e c tru m . From t h e p e n to se t e s t (T able I I I ) i t i s s e e n t h a t th e p e n to s e c o n te n t o f th e M irsky u n f e r t i l i z e d p r e p a r a t i o n wa3 a b o u t T h is i n d i c a t e d th e p o s s i b l e p re s e n c e of n u c l e i c a c i d . The m a t e r i a l was n e g a t iv e to t h e d ip h en y lam in e r e a g e n t o f D ische (1 9 3 0 ). T his would i n d i c a t e t h a t th e c o n s t i t u e n t was not d e s o x y r ib o s e n u c l e i c a c i d ; b u t M irsky and p o l l i s t e r (1946) s t a t e t h a t l a r g e q u a n t i t i e s o f p r o t e i n a r e known to i n t e r f e r e w ith th e r e a c t i o n . A n o th e r c r i t e r i o n f o r n u c le o p r o t e i n i s t h e a n a l y s i s o f t h e n i t r o g e n to p h ospho rus r a t i o (U:E) o f th e p r o t e i n . M irsky s t a t e s t h a t t h e t h e o r e t i c a l r a t i o f o r t h e sodium s a l t of d e s o x y r ib o s e n u c l e i c a c i d i s 1 .6 8 . From my d a ta i t i s s e e n t h a t t h i s r a t i o w ould be 121. 20 I would co n clu d e t h a t no l a r g e p o r t i o n o f th e M irsky p r o t e i n from u n f e r t i l i z e d eggs c o n s i s t s of n u c l e i c a c i d . 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 f u r t h e r , a b s o r p t io n t e s t s were r u n on t h e p r o t e i n u s i n g a Beckman Model D S p e c tr o p h o to m e te r . I t was o n ly p o s s i b l e to o b t a i n c u rv e s i n t h e u l t r a v i o l e t f o r th e M irsky p r o t e i n . T ra n s m is s io n was a lm o s t n i l th ro u g h th e u n f e r t i l i z e d and f e r t i l i z e d homogenate e x t r a c t s . I t is b e l i e v e d t h a t th e p igm ent echinochrom e i n t e r - f e r e d h e r e . A G.157 p e r c e n t s o l u t i o n o f M irsky p r o t e i n i n 0 .5 M KCl o f pH 7*0 was u s e d f o r th e t e s t s . Log 10 I 0/ l v a lu e s (where I 0= i n t e n s i t y o f th e i n c i d e n t l i g h t and l » t r a n s m i t t e d l i g h t ) were p l o t t e d d i r e c t l y a g a i n s t th e wave l e n g t h . E x t i n c t i o n c o e f f i c i e n t s were d e te rm in e d by th e B eer-L am bert e q u a t i o n : L ° e io I o / i E= ___________ c . d . w here E i s th e e x t i n c t i o n c o e f f i c i e n t , log^Q I 0/ l the d e n s i t y , c th e c o n c e n t r a t i o n and d th e d e p th of th e c e l l (1 c m .). The r e s u l t s a re shown i n F ig u re 1 . The main a b s o r p t i o n was from 258 th r o u g h 272 nju.. I n com paring i t w ith th e c u rv e s of M irsky and P o l l i s t e r (1946) f o r p u r i f i e d d e s o x y r ib o s e n u c l e i c a c i d and f o r Chrom osin (a n u c l e o p r o t e i n ) i t i s e v id e n t t h a t th e c l e a r c u t maximum a b s o r p t i o n a t 260 i s l a c k i n g . A s t e e p f a l l was n o te d a f t e r 272 nju. to 310 mp. The v a lu e f o r TABLE I I I CHEMICAL ANALYSIS OF THE EGG PROTEINS A ll Values R e la te d per ml. o f P r o t e in S o lu tio n % o f T o ta l P r o t e in as Phosphorus o f th e T o ta l Phosphorus % of T o ta l T o ta l T o ta l mg T o ta l P r o t e in mg P r o t e in mg as P r o t e i n N itro g e n N.x6-25 P entose Pentose T o ta l mg of Phosphorus mg o f Free Phosphorus % o f T o ta l as Free mg. o f Combined Phosphorus % of th e T o ta l as Combined "M irsky” P r o t e i n .586 Un f e r t i l i z e d 3.625 .112 3.09 .0327 .903 .0048 14.71 . 0279 85.29 F e r t i l i z e d E x t r a c t -753 4.707 .082 1.74 .0167 .356 .0040 23.08 .0127 76.92 Un f e r t i l i z e d E x t r a c t 1.128 7.050 .169 2.3 9 .0391 .555 .0068 17.46 .0322 82.54 ro ££ M irsky and p o l l i s t e r s Chromosin and n u c l e i c a c i d compound was £ 1 ,0 a t £60 njitt. ; mine f o r t h a t wave l e n g t h was 6 .0 1 , The c h a r a c t e r i s t i c minimum a t £30 nyx was n o t n o te d i n my p r e p a r a t i o n , i n g e n e r a l , i t can be s a i d t h a t t h e a b s o r p t i o n f o r th e M irsky p r o t e i n i s n o t s t r i c t l y c h a r a c t e r i s t i c o f a p r o t e i n o r n u c l e i c a c i d compound. T y p ic a l p r o t e i n c u rv e s a re i l l u s t r a t e d by M irsky and p o l l i s t e r and by L e rn e r and Barnum (1946) show ing a b ro ad maximum from £70 to £80 jjjU., and a minimum a t £50 mp. The minimum f o r th e M irsky p r o t e i n i s a t £5£ mji. , n o t f a r from th e above v a l u e . From th e d a t a on hand i t seems p o s s i b l e to s t a t e t h a t n u c l e i c a c i d i s n o t a m ajo r component a s th e maximum a t £60 njd. i s n o t d e f i n i t e and th e minimum of £30 to £40 njtt. i s n o t d e m o n s tr a te d . The c h e m ic a l a n a l y s i s , to o , does n o t i n d i c a t e any m ajo r p o r t i o n (BT:P r a t i o ) o f my p r e p a r a t i o n to be n u c l e i c a c i d . The m ajor p o r t i o n o f th e M irsky p r e p a r a t i o n i s u n d o u b te d ly p r o t e i n i n n a t u r e w i t h s m a ll c o n ta m in a tin g amounts of n u c l e i c a c i d and p o s s i b l y p ig m en ts from t h e o r i g i n a l e g g s. V i e s 1 and G-ex (19£8) have s t u d i e d th e u l t r a v i o l e t a b s o r p t i o n o f t h e mixed p r o t e i n s o f the u n f e r t i l i z e d p a r a c e n t r o t u s l i v i d u s egg. F or t h e i r p r e p a r a t i o n th e eggs were f r o z e n i n a l i q u i d a i r m ix tu re to d i s r u p t th e c e l l . They were th e n washed i n £ to 3 tim e s th e volume o f s e a w a te r and f i l t e r e d th ro u g h a swab o f c o t t o n . The f i l t r a t e was £3 f r o z e n i n a l i q u i d a i r m ix tu re and 0 .5 gram p o r t i o n s added to 10 m l. p o r t i o n s of a b u f f e r . These were th e n d i l u t e d 1 to 2 and th e sp e ctru m was m easured a t room t e m p e r a t u r e . A ssum ing the powdered p r o t e i n f i l t r a t e to be abo u t 80 p e r c e n t w a t e r , we would have a f i n a l c o n c e n t r a t i o n o f a b o u t 4 p e r c e n t . T h e ir sp e c tru m i s r e p l o t t e d a t I 0/ l and th e c u rv e s a t pHf s 7 .69 and 7 .7 1 a p p e a r i n F ig u r e £ a lo n g w i t h t h a t o f th e M irsky p r o t e i n from th e S t r o n g y l o c e n t r o t u s p u r p u r a t u s e g g . There was a b ro a d a b s o r p t i o n band from 250 t o £80 nyu. and th e g e n e r a l s lo p e o f th e c u rv e seemed to be q u i t e s i m i l a r to t h a t of the M irsky p r o t e i n . The Log^Q I 0/ i r e a d in g s and th e c o rr e s p o n d in g wave l e n g t h s a p p e a r i n T able IV f o r th e M irsky p r o t e i n and th e p r e p a r a t i o n of V ies and Gex. A T P -a se a c t i v i t y » The ATP u s e d i n t h e s e e x p e rim e n ts was p r e p a r e d a f t e r th e method o u t l i n e d by LePage i n "M anom etric T e c h n iq u e s - - ” (1 9 4 5 ). The p e rc e n ta g e o f l a b i l e p h o s p h a te ( r e a d i l y h y d ro ly z e d i n 1 K HC1 i n 15 m in u te s a t 100°C. and c o rr e s p o n d in g to £ m oles o f p h o sp h a te p e r mole o f ATP) was on th e o r d e r of 60^. A sam ple of ATP was o b ta in e d from Dr. Mehi l a t e r i n th e work ( p r e p a r e d a f t e r th e method o f K e r r , (1941) was o f t h e same o r d e r o f p u r i t y . The m o la r c o n c e n t r a t i o n s g iv e n a re c a l c u l a t e d on th e b a s i s o f t h i s 24 u j . n - y a u t a c > s o.r.p t i d n i f 7 » p ' w i p l s k y i !o. p &o teI n in— i n ; m o l a r . k «l ; N TFir :rt: D i 2»0 T .. . . . . . . . . . . . . . . . . . . . . . . . . . . . .-• ^ M — i SID 250 25 t t i i a A - vio u t a»soration op eU p t o m * iio 26 TABLE IV L°G10 I Q/ l VALUES FOR THE MIRSKY PROTEIH AED PROTEIHS OF VLES' & GEX AT VARIOUS ULTRA VIOLET WAVE LEJSGTHS Wave l e n g t h Log10 I 0/ l **o« l0 1 q/ 1 i n M irsky p r o t e i n P r o t e i n s o f v e ls» & Gex. PH 7 .0 pH 7 .69 pH 7 .7 1 230 1 .0 0 0 232 .9 7 0 235 .890 238 .790 240 .730 1 .0 0 0 244 1 .0 8 0 .730 .920 246 1.03 0 .900 248 1 .0 0 0 .720 .910 250 .960 252 .940 .720 254 .900 1 .0 0 0 256 .900 .760 1 .0 0 0 259 1 .0 0 0 260 .92 0 1.0 0 0 262 .920 .760 265 1 .0 0 0 270 .920 .7 5 0 1 .0 0 0 274 .900 275 .750 .97 0 276 .900 279 .730 280 .870 .725 .900 282 .840 286 .770 .550 .870 288 .750 289 .730 290 .67 0 .474 294 .56 2 .680 296 .500 298 .4 50 300 .408 .340 .600 310 .270 .250 .500 27 l a b i l e p h o s p h a te . H om ogenates. I t was f i r s t d e c id e d t o d e te rm in e the c o n c e n t r a t i o n o f t h e ATP-ase i n th e egg hom ogenates. p a p e r s by DuBois and p o t t e r (1 9 4 3 ). Mehi and S e x to n (1943) and S in g h e r and M e i s t e r (1945) were c o n s u l te d f o r m etho ds. The p ro c e d u re o u t l i n e d by Mehl was u s e d w i t h a few m o d i f i c a t i o n s . M easurem ents were made by a d d in g a l l of the com ponents to th e r e a c t i o n m ix tu re so t h a t t h e f i n a l volume would be 9 m l. a f t e r th e a d d i t i o n o f th e sa m p le . The i n c u b a t i o n was c a r r i e d ou t i n t e s t t u b e s immersed i n a w a te r b a th h e ld a t a c o n s t a n t te m p e r a tu re of 25°C. At th e c lo s e of 60 m in u te s th e r e a c t i o n was s to p p e d by a d d in g 1 m l. of 50 p e r c e n t t r i c h l o r a c e t i c a c i d . The c o n t e n t s o f th e tu b e were w e l l sh a k en and f i l t e r e d th r o u g h a Whatman pT o. 5 f i l t e r p a p e r . A l iq u o ts w ere th e n w ith d raw n from th e c l e a r f i l t r a t e and a n a ly z e d f o r p h o sp h o ru s a c c o rd in g t o th e method o f F is k e and Subbarrow (1 9 2 5 ). R eadings were made on t h e K le tt-S u m m erso n C o lo r im e te r u s i n g a 660 f i l t e r . T h i r t y m inute c o l o r developm ent p e r i o d s were a llo w e d and i t was fo u n d t h a t c a l i b r a t i o n c u rv e s were l i n e a r up to a c o n c e n t r a t i o n o f 40 gamma p e r 5 ml. I n a l l of the d e te r m i n a ti o n s s t a n d a r d s were c a r r i e d th r o u g h th e same p r o c e s s as f o r th e t e s t sam p le. I t was fo u n d t h a t th e c o l o r i m e t e r r e a d i n g s , h e r e , were th e same a s f o r t h o s e on th e 28 c a l i b r a t i o n c u r v e . I n w orking w ith th e p r o t e i n s o l u t i o n s the t e s t i n g p ro c e d u re was a l t e r e d som ewhat. H ere, th e r e a c t i o n m ix tu re s had a f i n a l volume o f 5 m l. a f t e r the a d d i t i o n o f the enzyme. The i n c u b a t i o n was a t 25°C. a s b e f o r e . At the c lo s e of 5 m inute p e r i o d s , 1 or 2 ml. sam ples were rem oved and d e l i v e r e d i n t o c e n t r i f u g e tu b e s c o n t a i n i n g an e q u a l amount o f 20 p e r c e n t t r i c h l o r a c e t i c a c i d . The sam p les were c e n t r i f u g e d a t 3500 R.P.M. f o r 20 m in u te s . One o r 2 m l. a l i q u o t s were w ith d ra w n from th e s u p e r n a t a n t and a n a ly z e d f o r in o r g a n i c p h o sp h o ru s as d e s c r i b e d e a r l i e r . The b u f f e r u s e d i n a l l t e s t s was sodium d i e t h y l b a r b i t u r a t e , ( n e u t r a l i z e d to pH 8 .5 w ith HC1) 0 .0 4 M. The m o l a r i t i e s a r e e x p r e s s e d as th e f i n a l r e a c t i o n c o n c e n t r a t i o n . DuBois and P o t t e r (1943) n o te d t h a t th e t i s s u e homo g e n a te s th e y d e s c r i b e d n eed ed a c a lc iu m a c t i v a t i o n f o r t h e ATP-ase p r e s e n t . The minimum c o n c e n t r a t i o n f o r c a lc iu m was 0.0007 7 M and no i n h i b i t i o n was n o te d above t h i s p o i n t . Magnesium, t o o , was fo u n d t o be a n a c t i v a t o r i n concen t r a t i o n s arou nd 0.00 15 M. I n c o n c e n t r a t i o n s above 0 .0 024 M magnesium i n h i b i t e d . I t was d e c id e d to r u n th e f i r s t t e s t s w i t h c a lc iu m and magnesium a c t i v a t i o n . The f i n a l r e a c t i o n m ix tu re m o l a r i t i e s w ere a s f o llo w s : 29 S u b sta n c e M o l a r ity 0 .0 400 B a r b i t u r a t e B u ff e r 0 .0 0 4 4 Mg 0.0017 ATP 0.0006 The c o n c e n t r a t i o n o f homogenate is e x p re s s e d i n mg. o f n i t r o g e n and the amount o f p h o sp h o ru s l i b e r a t e d a s o f p h o sp h o ru s p e r h o u r. The n i t r o g e n v a lu e s f o r th e f e r t i l i z e d homogenate ra n g e d from 0 .6 3 2 to 25.28 0 mg. and f o r th e u n f e r t i l i z e d homogenate - 0 .9 4 4 to 3 7 .7 6 0 mg. The r e s u l t s a r e shown i n f i g u r e 3. I t is i n t e r e s t i n g to n o te t h a t s i m i l a r v a lu e s were o b ta in e d i f t h e c a lc iu m and magnesium were n o t u s e d . We n o t e , t o o , t h a t t h e r e was l e s s n i t r o g e n i n th e f e r t i l i z e d hom ogenates. The g r e a t e r enzyme a c t i v i t y i n t h i s c ase may p e rh a p s be e x p la in e d by th e p r e s e n c e of more a c t i v a t o r s . P r e v io u s d a t a has shown t h a t the ash c o n te n t was much g r e a t e r i n f e r t i l i z e d e g g s . The d i f f e r e n c e cannot be e x p la in e d s a t i s f a c t o r i l y . The t r e a tm e n t f o r b o th s e t s o f eggs was i d e n t i c a l and th e o n ly p o s s i b l e e x p l a n a t i o n i s t h a t more s e a w a te r s a l t s were l e f t i n o n e . A p p ly in g th e c o r r e c t i o n f o r w a t e r c o n te n t we f i n d th e n i t r o g e n p e r mg. o f egg dry w e ig h t t o be a s f o l l o w s : 30 Egg Mg* IU p e r mg. Egg Dry W eight F e r t i l i z e d 0*0698 U n f e r t i l i z e d 0*1024 H utchens e t a l (1942) have d e te rm in e d th e H. value per mg. dry w e ig h t of th e A rb acia e g g . T h e ir mean was 0 .1 0 1 mg. f o r th e u n f e r t i l i z e d egg and i s in e s s e n t i a l agreem ent w i t h our v a lu e f o r th e S t r o n g y l o c e n t r o t u s u n f e r t i l i z e d egg. I n our t e s t s th e m icrogram s o f p h o sp h o ru s s p l i t p e r u n i t tim e w i l l be r e l a t e d d i r e c t l y to th e n i t r o g e n p r e s e n t i n the sa m p le . I n no c a s e s w ith th e hom ogenates o r th e p r o t e i n s o l u t i o n s d i d t h e enzyme s p l i t more th a n th e t e r m i n a l p h o sp h a te group of t h e ATP m olecule.. T h e r e f o r e , d a t a w i l l be r e p o r t e d i n p e rc e n ta g e o f th e t e r m i n a l p h o sp h a te o f ATP h y d r o ly z e d . The v a lu e s f o r the hom ogenates a re r e p o r t e d i n t a b l e V. The r e s u l t s a ls o a p p ea r i n g ra p h form i n f i g u r e 3. F e r t i l i z e d and u n f e r t i l i z e d e x t r a c t s . The t e s t i n g p ro c e d u re u s e d i n t h i s ease has been d e s c r i b e d i n d e t a i l e a r l i e r . The f i n a l r e a c t i o n m ix tu re volume was 5 m l. a f t e r th e a d d i t i o n of th e enzyme and i n c u b a t i o n was f o r 1 ho ur a t 25°C* At th e c lo s e of th e a p p o in te d tim e - 2 ml. a l i q u o t s were p i p e t t e d from t h e t u b e s and d e l i v e r e d i n to 2 ml. p o r t i o n s o f 20^ t r i c h l o r a c e t i e a c i d i n 15 m l. c e n t r i f u g e t u b e s . These were c e n t r i f u g e d ard 2 ml. p o r t i o n s u se d f o r TABLE V HYDROLYSIS OP ATP BY THE FERTILIZED AND UNFERTILIZED EGG HOMOGENATES Homogenate U n f e r tiliz e d . F e r t i l i z e d M illigram s o f N itro g en in Sample Microgram o f Phosphorus s p l i t per hour Per c en t o f the T erm inal ATP-P Hydrolyzed .94 3-9 2 .2 9 3.77 9-0 5 .29 4.72 15.1 8.88 9.44 2 2 .6 13.29 1 8 .88 55.6 32.70 28.32 82.7 48.64 37.76 111.2 65.41 .63 6 .5 3-82 2 .52 13.8 8 .1 1 3.16 2 2 .9 13.47 6.32 41.2 24.23 12.64 7 6 .9 45.23 1 8 .96 121.4 71.41 25.28 153.8 90.47 32 e e e e i i __ St ... ' ' 1 ........r ........p - r P I 6 U R. E 3 . 1TY OF SEA URCHfN E 6 6 r- 1— ' . . — - H I * T P r - - j — 1 -■ - 4 ■ - ■ ■ l XCTIV ' T “ ~ MOMOG L T lS 1 ■ .'■ i ; 1 s .. . HE. ACT IOM M l Y T U J t t ( S h v i . V O L U M t, - - - T -- » ! 0 .6 0 4 4 M M t D f > i i i --------- o . o o n BOLOfL N MOUkfl 4 fla*4 . 1. .; -— j | 0 . 0 0 0 6 k T P j r t . f — ------- BO MINUTE INCUMTIOI SI, Z 5 * e i i r f J r : ! ; 1 5 ■ ; .------- . • .1 — r .._ " I -------- ----7 -._ . r -------- ID0 i • --■i---- ---- 1 t ( ■ ) ItJV j i ; • • — — — ■ /; — F SJL 0 .0 * n u i t B U 6 < i BO- m f l u I . - . : i wu ■ f r i 1 1 Jll —- — r— — • — — ? - ■ ■ '• / 1 — ■ • — ! -- lfc« --- ----- - __j_. - ' — ,X i • j • i XT ' X ; i . L . j l . — i . • - -A - — • • - ' * • - • . i : / r f ; : ■ :. / / i $ * "\".A / -i • T i OA i y : --- ■ ■ -} .-4, ; < b --7 --- > ; — — . - — j— ' V r iia a KOt_ 1 AOS - X 'l . .. fe * & » « t / 66i i i • ---- w - . I . X y * ..j.. - i : } ■ ■ ■ ' :j. ' ■ T T T t 7 - : | ! i ■y % • ! • r j ;; . i j ; 2 “ y * ! i - ■ ' . ! ' i ' . . !.l.... r:-: : . j.-:. i ■ ■ -rv ; < . • . . . v y * • • • 1 .. I..: ... i ;;;; j : 9J r * / / j- _ - -tv 1 - 'i : rr-r 1 - 1 • X in : X X : : ----' —f t —TO _ _ ; x • P . —----- i ■ : : i - ■ | — 1 - 4 - — — • T X - y x . ■ i - " i - i A i ........ “1 ---- TO 'JT s ! ■ ; r • * :i * ; ' j f > .. L. i i • ! — n r . / y i — : — — — p . . . . : — - - . . . . . | # ...........i ...... . j . • I 1 M l > IB j BO • SB L L I G I t A M S OF N I T i p Q E M . 1o : 1 » : ' " T I b, . . . . . . . .j . . . . . ! ! ----------------- : j - i i i.” 33 th e p h o sp h o ru s d e t e r m i n a t i o n . S ta n d a rd s were c a r r i e d t h r o u g h 't h e same p ro c e d u re and b l a n k s on r e a g e n t s , sam ple and ATP were s u b t r a c t e d from th e f i n a l v a l u e s . C alcium and magnesium were added b o th t o g e t h e r and s e p a r a t e l y to t e s t t h e i r a c t i o n . The f i n a l r e a c t i o n m ix tu re m o l a r i t i e s w ere: S u b sta n c e M o l a r it y B a r b i t u r a t e B u f f e r 0.04000 C a ++ 0.05000 ■+ ■ + ■ Mg 0.05000 ATP 0.00054 The n i t r o g e n l e v e l s on th e f e r t i l i z e d an d u n f e r t i l i z e d e x t r a c t s were 1.506 mg. and £.256 mg. r e s p e c t i v e l y . I n T able V th e amount o f ph o sp h o ru s l i b e r a t e d p e r h o u r and th e p e rc e n ta g e h y d r o l y s i s o f t h e t e r m i n a l p h o sp h a te o f ATP i s r e l a t e d to th e io n s add ed to the r e a c t i o n m ix tu r e : D e f i n i t e magnesium i n h i b i t i o n was shown i n t h e s e r e s u l t s . C alcium had an a c t i v a t i n g e f f e c t an d somewhat o f f s e t the i n h i b i t o r y e f f e c t of magnesium. On th e b a s i s o f mg. o f n i t r o g e n th e c a lc iu m a c t i v a t e d e x t r a c t s l i b e r a t e d th e f o llo w i n g gamma o f p h osp horus p e r h o u r; F e r t i l i z e d ----------- 6 .6 4 m icrogram s P . / h r . / m g . H. U n f e r t i l i z e d 9 .59 m icrogram s P . / h r . / m g . H. 34 TABLE VI THE EFFECT OF CALCIUM AND MAGNESIUM IOH Oil THE ATP-aae ACTIVITY OF THE FERTILIZED AND UNFERTILIZED EGG PROTEIN EXTRACTS E x t r a c t i o n added M icrogram s o f p h o sp h o ru s h y d ro ly z e d p e r h o u r p e r c e n t a g e o f T erm in al ATP-p h y d ro ly z e d U n f e r t i l i z e d C alcium 2 1 .6 5 25 .77 C alcium and magnesium 1 8 .3 0 21.38 Hone 1 2 .3 0 1 4 .4 0 Magnesium 0 . 0 . C alcium 10 .0 0 11 .9 0 C alcium and magnesium 6 .4 0 7 .6 1 Hone 5 .0 0 5.95 Magnesium 0 . 0 . 35 An a c t i v i t y t e s t a g a i n s t tim e was r u n on th e u n f e r t i l i z e d e x t r a c t a c t i v a t e d w ith c a lc iu m . The same pH and co n cen t r a t i o n o f b u f f e r and s u b s t r a t e w ere u se d a s b e f o r e . The l i b e r a t i o n o f phosp horu s was l i n e a r th ro u g h a p e r i o d of tw e n ty m in u te s . 2 .2 5 6 mg. o f B. l i b e r a t e d th e f o l l o w i n g M icrogram of P h o s p h o ru s • Time M icrogram o f P h o sp h o ru s 2 Min. 1.65 5 « o o • 10 " 8 .0 5 15 " 1 1 .8 0 20 1 1 1 5 .0 0 25 1 1 1 7 .0 5 30 » 18.8 5 The r e s u l t s a p p e a r i n f i g u r e 4 . The M irshy p r o t e i n . T his f r a c t i o n did n o t p o s s e s s any ATP-ase a c t i v i t y r e g a r d l e s s o f th e so u rc e of e g g s. T e s ts were ru n w i t h and w ith o u t c a lc iu m a c t i v a t i o n and a l s o w ith v a r y i n g th e s u b s t r a t e and p r o t e i n c o n c e n t r a t i o n . I t was con c lu d e d t h a t th e ATP-ase d e m o n s tra te d i n th e e x t r a c t d i d no t come from t h i s com ponent. M yosin. A sam ple o f r a t m yo sin , p r e p a r e d a f t e r th e 56 0.M00 3 M D L A R . bftiM TtHlATfi MF'tR. J^a&ooa. MftLAB-— c* — i l 0 . 0 0 0 : 4 MOLAK ATP i PI.OX Cl M N . - 0 . 4 * 1 2 » n u / » n l ; -ik- 25 37 m ethod of S in g h e r and M e is te r (1945) was t e s t e d f o r i t s ATP- a se a c t i v i t y as a check on th e m ethod o f a s s a y . A h y d r o l y s i s c u rv e o f ATP a g a i n s t tim e was o b t a i n e d . The c o n c e n t r a t i o n o f k . i n th e t e s t sample was 0 .2 3 4 m g ./m l. and th e m o l a r i t i e s o f th e r e a c t i o n m ix tu re com ponents a r e l i s t e d below ; S u b stan c e M o la r ity B a r b i t u r a t e B u f f e r 0 .04000 Ca++ 0 .0 1 0 0 0 ATP 0 .0 0 0 5 2 The t e s t i n g p ro c e d u re was t h e same a s u s e d f o r th e u n f e r t i l i z e d and f e r t i l i z e d e x t r a c t s . The m ierogram s of p h o sp h o ru s h y d ro ly z e d p e r u n i t tim e and th e p e r c e n ta g e o f t e r m in a l p h o sp h o ru s h y d ro ly z e d from th e ATP s u b s t r a t e a re l i s t e d below; M icrogram s of P. p e r c e n t o f T erm in a l Time i n M inutes__________ h y d ro ly z e d ___________ ATP-P h y d ro ly z e d 1 2 6 .1 4 3 .5 0 5 4 9 .6 8 2 .6 6 15 5 6 .5 9 4.1 6 30 5 9 .3 9 8 .8 2 The r e s u l t s a re shown i n F ig u r e 5. I n c o n c lu d in g th e ATP-ase t e s t s on th e hom ogenates, M irsky p r o t e i n s , and f e r t i l i z e d and u n f e r t i l i z e d e x t r a c t s i t can be s t a t e d t h a t only t h e M irsky p r o t e i n s f a i l e d to show enzyme a c t i v i t y . The r e s u l t s a r e sum m arized i n a c h a r t 38 UTI' C T J V IT Y » I N ITm T N uam) rain 40iiA W ft* p 4 I M I O 39 below i n w hich th e enzyme a c t i v i t y i s e x p r e s s e d a s m icrogram o f p h o sp h o ru s h y d ro ly z e d from ATP p e r hour p e r m il lig r a m o f n i t r o g e n i n th e enzyme m a t e r i a l . S u b sta n c e M icrogram p . / h r . / m g . K. M irsky p r o t e i n s from : U n f e r t i l i z e d eggs 0 F e r t i l i z e d eggs 0 F e r t i l i z e d egg homogenate 10.28 U n f e r t i l i z e d egg homogenate 4 .1 3 F e r t i l i z e d egg e x t r a c t 6 .6 4 U n f e r t i l i z e d egg e x t r a c t 9.59 B ~ G ly ce ro p h o sp h a ta se a c t i v i t y . There e x i s t e d th e p o s s i b i l i t y t h a t an a l k a l i n e p h o s p h a ta s e was p r e s e n t i n th e hom ogenates and e x t r a c t s c a p a b le o f s p l i t t i n g th e l a b i l e p h o sp h a te groups of ATP. T e s ts were c a r r i e d out u s i n g Sodium B G ly c e ro p h o sp h a te as a s u b s t r a t e and k e e p in g o t h e r f a c t o r s s i m i l a r t o th o s e i n th e ATP-ase w ork. 4 B -g ly c e r o p h o s p h a ta s e a c t i v i t y was d e m o n s tra te d i n th e hom ogenates. However, 20 h o u r s 1 i n c u b a ti o n was needed to p rodu ce ab out 37 p e r c e n t h y d r o l y s i s . A t e s t u s i n g a u n f e r t i l i z e d egg homogenate c o n t a i n i n g 4 .7 2 mg. o f E. i s r e p o r t e d . Mg"1 "* was u se d a s an a c t i v a t o r . 40 S u b s ta n c e M o la r ity B a r b i t u r a t e B u f fe r 0.04 0 0 0 MgT+ 0.01000 S o d iu m -B -G ly cero p h o sp h ate 0.00 634 Time o f Gamma of P . $ o f T o ta l P . I n c u b a t i o n H y d ro ly zed H yd ro lyzed 1 h o u r 7 .1 3 .6 1 20 h o u rs 74*3 3 7.79 As no a c t i v i t y was o b t a i n e d i n 1 h o u r s 1 tim e w ith th e M irsky p r o t e i n or e x t r a c t s , i t was c o n c lu d e d t h a t t h i s p h o sp h a ta s e a c t i v i t y was n o t c o n ta in e d i n them. I t i s o b v io u s t h a t f r a c t i o n p o s s e s s i n g ATP-ase a c t i v i ty i s l o c a t e d somewhere i n th e u n f e r t i l i z e d e x t r a c t . I t m ig h t be 1 o r more o f th e v i s i b l e components bu t one co u ld n o t be d e f i n i t e on t h i s p o i n t . S in ce t h e ATP-ase d e m o n s tra te d was r a t h e r weak, th e p o s s i b i l i t y e x i s t s t h a t th e a c t i v e enzyme was a d s o rb e d on one of t h e com ponents. A c a r e f u l f r a c t i o n a t i o n ( i f p o s s i b l e ) would have to be made on eac h o f th e com ponents, and th e n enzym atic t e s t s to c l a r i f y t h i s p o s s i b i l i t y . I t i s f e l t t h a t th e homogeneity of th e M irsky p r o t e i n has been d e m o n s tr a te d by th e s e t e s t s , and a l s o th e f a c t t h a t a t l e a s t f o u r com ponents w ere e x t r a c t e d w ith G.6 M KCl o f 41 pH 8 .5 from th e s e a u r c h i n egg. ELECTROPHORESIS The M irsky p r o t e i n has been r e f e r r e d to as a s i n g l e f r a c t i o n and t h a t a c t i o n has b e en j u s t i f i a b l e i n t h e l i g h t o f e l e c t r o p h o r e t i c a n a l y s i s . However, t e s t s on th e u n f e r t i l i z e d e x t r a c t p o s s e s s i n g ATP-ase a c t i v i t y have r e v e a l e d 4 m a jo r com ponents, the l a r g e s t c o m p ris in g some 4 0^ o f th e t o t a l p r o t e i n . The a u th o r i s i n d e b t e d to Dr. John W. Mehl and Mrs. Ja n e Humphrey f o r th e e l e c t r o p h o r e t i c s t u d i e s r e p o r t e d . The p r o t e i n s were made up i n a v e ro n a l-K C l b u f f e r of th e f o l lo w in g c o m p o s itio n : 0 .0 2 M. sodium d i e t h y l b a r b i t u r a t e , 0 .0 8 M. KC1 and n e u t r a l i z e d to pH 8 .5 w i t h cone HC1. To i n s u r e e x a c t m o l a r i t i e s the p r o t e i n s were th e n d i a l y z e d a g a i n s t s e v e r a l s m a ll sam ples of th e same b u f f e r and f i n a l l y a g a i n s t a l a r g e p o r t i o n f o r two d ay s b e fo re th e e l e c t r o p h o r e t i c r u n . i n t h e ease of the u n f e r t i l i z e d e x t r a c t th e c o n ce n t r a t i o n of th e p r o t e i n s was 0*705^. The M irsky p r o t e i n had t o be made i n a c o n c e n t r a t i o n of Q.G4j|. I n b o th c a s e s th e m i g r a t i o n was s w i f t , l a s t i n g f o r only 2 h o u rs . The u n f e r t i l i z e d e x t r a c t components were i n th e f o l l o w i n g o r d e r o f m ag n itu d e: 4 2 1 - 7.77j£ These v a lu e s were f o r th e a s c e n d in g 2 - 4 0 . 0 0 portion, th e d e c e n d in g was much th e 3 - 2 7 .7 7 $ same. 4 - 2 4 .4 7 $ The p a t t e r n s o b ta in e d w i t h b o th a s c e n d in g b o u n d a rie s a re shown i n P l a t e s I I and I I I . DOUBLE REFRACTION OF FLOW (DEF) The p rim a ry p u rp o se f o r t h i s t e s t was t o check on th e p o s i t i v e b i r e f r i n g e n c e r e p o r t e d by M irsky f o r h is p r o t e i n . I n r e c e n t y e a r s many p h y s i c a l methods have been d e v e lo p e d f o r th e c h a r a c t e r i z a t i o n o f th e shape and s i z e o f p r o t e i n s . As Mehl (1938) has s t a t e d , th e m easurem ent o f d o u b le r e f r a c t i o n of flo w has p ro v id e d a -useful su p p lem en t f o r t h e s e e x i s t i n g m ethods more g e n e r a l l y i n u s e . The r e a d e r i s r e f e r r e d a g a i n to th e e x c e l l e n t a r t i c l e o f E d s a l l ^ i n Volume 1 o f "R ecent Advances i n C o l lo i d Science*1 f o r a c r i t i c a l t r e a t m e n t o f t h i s phenomenon. Also to E d s a l l e t a l. (1944) f o r a d e s c r i p t i o n of a p r e c i s e in s tr u m e n t f o r m ea su rin g the amount o f d o u b le r e f r a c t i o n o f flow a t h ig h v e l o c i t y g r a d i e n t s . Mehl (1938) has d e s c r i b e d a t l e n g t h t h e ap p l i c a t i o n s of t h i s m ethod i n the f i e l d of p r o t e i n c h e m is tr y . The b a s i c p r i n c i p l e s in v o lv e d i n t h i s p ro c e d u re w i l l 43 ELECTROPHORETIC ASCENDING- BOUNDARY OF THE UNFERTILIZED EGG PROTEINS PLATE I I I ELECTROPHORETIC ASCENDING BOUNDARY OF THE-UNFERTILIZED EGG KERSEY PROTEIN 45 be d i s c u s s e d b r i e f l y . O rd in a ry s u n l i g h t o r room l i g h t v i b r a t e s i n a l l p l a n e s . When one p a s s e s a beam of l i g h t th r o u g h a H ic o l p ris m o r p ie c e of P o l a r o i d g l a s s , i t becomes p la n e p o l a r i z e d . We mean by t h i s t h a t i t i s now v i b r a t i n g i n one p l a n e . I f a second H ic o l p ris m i s p l a c e d so t h a t i t s p la n e o f p o l a r i z a t i o n i s a t r i g h t a n g le s t o th e f i r s t , t h e n th e l i g h t w i l l be e x ti n g u i s h e d by th e l a t t e r . One may p la c e a c r y s t a l , eg. c a l e i t e betw een th e c r o s s e d H ie o ls and th e l i g h t i n p a s s i n g th r o u g h t h i s m a t e r i a l w i l l become e l l i p t i c a l l y p o l a r i z e d . We w i l l n o t be a b le to e x t i n g u i s h t h i s l i g h t any lo n g e r no m a t t e r i n w hat d i r e c t i o n we r e v o lv e th e seco n d H ic o l. S u b s ta n c e s c a p a b le o f a l t e r i n g p la n e p o l a r i z e d l i g h t i n su c h a m anner a re term ed o p t i c a l l y a n i s o t r o p i c or d o u b le r e f r a c t i v e . Those which do n o t a l t e r i n t h i s r e s p e c t a re c a l l e d i s o t r o p i c . We m ight c o n s i d e r a s u s p e n s io n or s o l u t i o n o f l o n g r o d - l i k e a n i s o t r o p i c p a r t i c l e s . I f th e y were i n random o r i e n t a t i o n , th e n th e o p t i c a l e f f e c t s o f any one w ould be c a n c e l l e d by th e o t h e r s . However, i f i t were p o s s i b l e to o r i e n t them so t h e i r o p t i c a l a x es were p a r a l l e l , th e n th e e f f e c t sh o u ld be as f o r 1 l a r g e a n i s o t r o p i c p a r t i c l e . We f i n d t h a t t h i s i s th e c a se an d c a n be d e m o n s tra te d by s t i r r i n g su c h p a r t i c l e s i n a s o l u t i o n so t h a t t h e i r lo n g a x e s a re 46 o r i e n t e d i n th e d i r e c t i o n o f th e s tr e a m l i n e s o f t h e f l u i d * T his i s th e fu n d a m e n ta l p r i n c i p l e o f d o uble r e f r a c t i o n o f flow* There a r e a few v a r i a t i o n s and e x c e p t i o n s t h a t a r e t a k e n up i n th e r e f e r e n c e s m entioned* F o r th e work i n t h i s r e s e a r c h a s m a l l d e m o n s tr a tio n a p p a r a tu s was o b t a i n e d from Dr* Mehl. I t c o n s i s t e d of a f i x e d o u t e r c y l i n d e r and a movable i n n e r c y l i n d e r so con s t r u c t e d as t o move by a s m a ll b e l t pow ered by an e l e c t r i c motor* The s p e e d o f th e m otor c o u ld be c o n t r o l l e d by a r h e o s t a t . The o u t e r c y l i n d e r was o f b r a s s w ith a g l a s s p l a t e on th e b o tto m ; th e i n n e r c y l i n d e r was o f l e u c i t e and moved f r e e l y i n i t s m ount. P o l a r o i d d i s c s were p la c e d i n a c r o s s e d p o s i t i o n above and below th e ends o f th e a p p a r a tu s and a l i g h t s o u rc e came from below th e 1 s t . P o l a r o i d . The s o l u t i o n t o be t e s t e d was p la c e d i n th e a n n u la r sp a ce b e tw een th e two c y l i n d e r s and th e f i e l d o f th e P o l a r o i d n o te d , i f th e s o l u t i o n p a r t i c l e s were a t random o r i e n t a t i o n , th e n the f i e l d a p p e a re d d a rk . However, i f th e m ix tu re was a g i t a t e d by r e v o l v in g th e i n n e r c y l i n d e r , a r a t e of s h e a r was s e t up o r i e n t a t i n g th e p a r t i c l e s p a r a l l e l to th e s tr e a m l i n e s of th e l i q u i d . The l i g h t was n ot e x t i n g u i s h e d by th e seco nd P o l a r o i d and the s o l u t i o n was term ed d o u b le r e f r a c t i v e . Such a t e s t was c a r r i e d o u t on th e M irsky p r o t e i n from u n f e r t i l i z e d e g g s . 47 I n P l a t e s IV, V and V I, p h o to g ra p h s a p p e a r o f th e a p p a r a t u s u s e d . I n H a t e IV a view o f th e a sse m b le d machine i n o p e r a t i o n i s shown. P l a t e V i s a c lo s e - u p view of t h e c y l i n d e r s and P l a t e VI i s an " e x p lo d e d 1 1 p i c t u r e o f th e c y l i n d e r s and o p t i c a l a p p a r a t u s . The p o l a r o i d s a re a t th e e xtrem e ends i n t h i s l a s t P l a t e , P l a t e s VII and V III were p h o to g ra p h s o f th e M irsky p r o t e i n b e fo r e and d u r i n g a g i t a t i o n i n th e a p p a r a t u s . I t c a n be c l e a r l y se e n t h a t th e f i e l d ( n e x t to t h e i n n e r c y l i n d e r ) i s a n i s o t r o p i c when th e c y l i n d e r s a r e i n m o tio n . The p r o t e i n c o n c e n t r a t i o n was 1 .1 2 ^ . The phenomena o f DBF was n o t n o te d i n t h e u n f e r t i l i z e d a n d f e r t i l i z e d e x t r a c t s , p ro b a b ly b eca u se o f t h e lo w e r c o n c e n t r a t i o n . E a r l i e r t e s t s had i n d i c a t e d some b i r e f r i n g e n c e , b u t th e phenomenon was q u i te weak and n o t e a s i l y p h o to g ra p h e d . As p o i n t e d o u t p r e v i o u s l y , t h e f i e l d a p p e a rs d a rk b e f o r e th e r o t a r y m otion o f th e c y l i n d e r b u t b r i g h t d u r i n g t h e p r o c e s s . I n P l a t e V III we s e e a d a rk c r o s s , th e a r a s o f w hich a r e a t 90° from e a c h o t h e r . T h is i s c a l l e d th e "C ross o f I s o c l i n e " and i s u se d to some e x t e n t i n m e a s u rin g th e d e g re e of b i r e f r i n g e n c e o f s o l u t i o n s . A d i s c u s s i o n o f t h i s phenomenon w i l l n o t be a tt e m p t e d h e r e . I n making e x tre m e ly a c c u r a t e m easurem ents o f DRF, a c c o u n t must be made f o r th e t e m p e r a t u r e , pH of t h e s o l u t i o n , 48 c o n c e n t r a t i o n o f p a r t i c l e s , and m o l a r i t y of d i s s o l v e d s a l t i n c a se o f p r o t e i n s ) , i n our t e s t t h e pH was 7 .0 , KCl con cen t r a t i o n was 0.5M, 1 .1 2 $ s o l u t i o n u s e d , and t h e t e s t s c a r r i e d o u t a t o r d i n a r y room te m p e r a tu r e ( n e a r 2 5°C ), and an R.p.M* o f a b o u t 100. ELECTRON MICROGRAPHS A s e r i e s of e l e c t r o n m ic ro g ra p h s were k i n d ly made on th e u n f e r t i l i z e d M irsky P r o t e i n by D r. D a n ie l p e a s e . D r. P e a se has been e x p e r im e n tin g w ith e l e c t r o n m ic ro sc o p e s t u d i e s o f m yosin and o t h e r f i b r o u s p r o t e i n s f o r th e p a s t y e a r and a h a l f . He has d e v e lo p e d a s p e c i a l f r e e z i n g and l y o p h i l e t e c h n iq u e f o r f i x i n g t h e p r o t e i n m a t e r i a l s u n d e r stu d y and to m inim ize th e p o s s i b i l i t y o f d i s r u p t i n g m i e e l l a r p a r t i c l e s . O th e r w o rk e rs i n th e f i e l d have m ain ly u s e d a c i d f i x a t i v e m ethods i n th e s tu d y o f p r o t e i n m a t e r i a l u n d e r t h e e l e c t r o n m ic ro s c o p e . The main purp ose i n t a k i n g t h e s e p h o to g ra p h s was to s e e i f any s t r u c t u r a l rese m b le n c e t o m yosin was shown by th e M irsky p r o t e i n . The t a s k o f i n t e r p r e t i n g th e r e s u l t s was c a r r i e d o u t by D r. P ease who c a n q u a l i f y a s an e x p e r t . No c la im s a re made by me a s to any s p e c i f i c s i m i l a r i t y betw een th e two p r o t e i n s . D r. P ease has r e p o r t e d t h a t f i b r o u s 49 PLATE IV PHOTOGRAPH OF THE ASSEMBLED DOUBLE r REFRACTION OF LOW APPARATUS 1 - Mo t a r Z r D r iv e b e l t 3 - O u te r c y l i n d e r 4 - P o l a r o i d „ 1 . 50 3 . PLATE V . VIEW OF THE ASSEMBLED POLAROIDS APPROXIMATELY ORE-HALF ACTUAL SIZE I - P o l a r o i d Z - i n n e r c y l i n d e r 3 ~ O u te r c y l i n d e r 4 ~ Fram e 53. PLATE YI "EXPLODED” VIEW OF THE CYLINDERS AND OPTICAL ATTACHMENTS 1 - P o l a r o i d s 2 * I n n e r C y lin d e r , 5 - O u te r C y lin d e r PLATS V II PHOTOGRAPH OF THE MIRSKY PROTEIH FIELD BEFORE AGITATIOH 1 - I n n e r C y lin d e r Z - O u te r c y l i n d e r W a ll 3 - D a rk e n e d F i e l d PLATS V I I I PHOTOGRAPH OF THE MIRSKY PROTELH FIELD DURING AGITATIOH 1 - i n n e r C y lin d e r 2 - O u te r C y l in d e r W a ll 3 - A n i s o t r o p i c F i e l d 4 - C ro s s o f I s o c l i n e 54 p r o t e i n s e g . c o l l a g e n , show a m ark ed ly d i f f e r e n t p i c t u r e th a n m y o sin . When more d a ta and i n f o r m a t i o n can be c o l l e c t e d i n th e f u t u r e on th e n a tu r e o f p r o t e i n s u n d e r th e e l e c t r o n m ic ro s c o p e , i t i s b e l i e v e d t h a t t h i s i n s tr u m e n t w i l l become an im p o r ta n t t o o l i n p r o t e i n c h e m i s t i y . R a b b it m yosin was p r e p a r e d by D r. P ease a f t e r th e m ethod o f S in g h e r and Me i s t e r . The f i n a l p r e p a r a t i o n was made up i n 0 .6 M. KC1. The M irsky u n f e r t i l i z e d p r o t e i n was th e same a s u s e d f o r t h e u l t r a v i o l e t a b s o r p t i o n w ork. The f i n a l c o n c e n t r a t i o n was 0*157$ and i t was d i s s o l v e d i n 0.5M KC1 o f pH 7 . 0 . D ata i s n o t a v a i l a b l e f o r th e concen t r a t i o n o f pH o f P e a s e s ’ p r e p a r a t i o n . The n e x t 2 p l a t e s a re p h o to g ra p h s o f m yosin and the M irsky p r o t e i n . P l a t e IX i s D r. p e a s e s ’ p r e p a r a t i o n o f m yosin m a g n if ie d 75,000 X. P l a t e X i s my p r e p a r a t i o n of th e M irsky P r o t e i n m a g n ifie d 75,000 X. The s m a ll , f a i r l y u n i form d o t s o f g r e a t e r e l e c t r o n d e n s i t y a r e e v id e n t i n e a c h e a s e . C r i t i c a l m easurem ents have been made by p e a se on t h e m yosin ’’d o t s ” and i t i s s t a t e d t h a t th e y have a f a i r l y o c o n s t a n t d ia m e te r o f a b o u t 2 6 0 A. C om pu tatio ns b a sed on a m o le c u la r w e ig h t of 1 m i l l i o n c o u ld r e l a t e t h e s e p a r t i c l e s to i n d i v i d u a l m o le c u le s o f m yosin, a c c o r d i n g to p e a s e . S i m i la r d o t s a re n o te d i n th e p i c t u r e s of th e M irsky P r o t e i n b u t i t i s s t a t e d t h a t t h e i r d ia m e te r i s n ot veiy 55 c o n s t a n t . The r e s u l t i s n o t r e p o r t e d a s o n ly a few m e a su re m ents were t a k e n . These p h o to g ra p h s a r e i n c l u d e d m ainly f o r th e i n t e r e s t t h a t th e y convey. I t seems t h a t p a r t i c l e s a re d e m o n s tr a te d i n each c a se w hich m ight he m o le c u le s . They a p p e a r c y l i n d r i c a l and n o t rod s h a p e d . 56 i iii PLATE IX ELECTROS MICROGRAPH OF RABBIT MTOSIH* 7 5 ,0 0 0 X i n ,6m KOI pH 7 ,4 57 PLATE X ELECTRON MICROGRAPH OP THE MIRSKY PROTEIN PROM UNFERTILIZED EGGS 7 5 ,0 0 0 X 0 .1 5 $ P e r c e n t p r o t e i n i n 0.5M KC1, pH 7 .0 CHAPTER I I I CONCLUSIONS AND DISCUSSION The p r o t e i n w h ic h u n d e rg o e s a s o l u b i l i t y d e c r e a s e d u r i n g th e p r o c e s s o f f e r t i l i z a t i o n h a s b e e n i s o l a t e d from f e r t i l i z e d and u n f e r t i l i z e d s e a u r c h i n e g g s , t h u s c o n firm i n g th e work of M irsky, Homogenates were p r e p a r e d fro m b o th f e r t i l i z e d an d u n f e r t i l i z e d eggs and fo u n d to be p o s i t i v e i n r e s p e c t to ATP-ase a c t i v i t y . P e r mg, o f N* the f e r t i l i z e d hom ogenates w ere more a c t i v e , b u t th e p re s e n c e o f a g r e a t e r p e rc e n ta g e o f a s h i n th e eggs s u g g e s te d th e p o s s i b i l i t y of m e ta l a c t i v a t i o n . When e x t r a c t s o f th e hom ogenates were t e s t e d th e u n f e r t i l i z e d e x t r a c t p ro v ed to be more a c t i v e p e r mg. o f N, t h a n were s i m i l a r e x t r a c t s from f e r t i l i z e d e g g s . The M irsky p r o t e i n was t e s t e d c h e m ic a lly and s p e c t r o - p h o t o - m e t r i c a l l y and t h e d a t a on hand do n o t w a rre n t the c o n c l u s i o n t h a t any m ajo r p o r t i o n o f i t i s n u cleop ro 'frein. The u s u a l N:P r a t i o of n u c l e o p r o t e i n s i s 1 .6 8 and t h i s m a t e r i a l had a r a t i o of 1E1. A b s o r p tio n by u l t r a v i o l e t l i g h t d id n o t e x h i b i t th e c h a r a c t e r i s t i c maximum a t E60 njh. n o r th e minimum a t E50 njjui. common f o r n u c l e i c a c id compounds. I t was c o n c lu d e d t h a t a p r o t e i n was p r e s e n t c o n ta m in a te d to some e x t e n t by n u c l e i c a c i d . The M irsky p r o t e i n was con s i d e r e d to be a s i n g l e f r a c t i o n from the r e s u l t s of 59 e l e c t r o p h o r e s i s t e s t s . S tre a m in g DRF was d e m o n s tra te d s u b s t a n t i a t i n g M irsky* s c la im . ATP-ase t e s t s w ere n e g a t i v e . A d e c r e a s e i n p r o t e i n s o l u b i l i t y d u r i n g f e r t i l i z a t i o n was c l e a r l y shown when one compared th e p r o p o r t i o n s o f n i t r o g e n e x t r a c t e d from f e r t i l i z e d and u n f e r t i l i z e d eggs by the M irsky m ethod. Only 9 .0 1 p e r c e n t o f th e t o t a l n i t r o g e n was e x t r a c t e d from f e r t i l i z e d eggs b u t 1 6 .3 3 p e r c e n t i n th e case o f u n f e r t i l i z e d e g g s . The v a lu e s h e re were n o t a s h ig h as M irsk y * s c la im , b u t he was s u b s t a n t i a t e d i n p r i n c i p l e . The ATP-ase from th e Weber S o l u t i o n e x t r a c t s was a c t i v a t e d by Ca++ and i n h i b i t e d by Mg**" i n 0 .0 1 M concen t r a t i o n s . The minimum c o n c e n t r a t i o n s were n o t t e s t e d due to s h o r t a g e of p r o t e i n m a t e r i a l . However, th e e f f e c t o f Mg^+ was p a r t l y o f f s e t by Ca . Only t h e t e r m i n a l l a b i l e p h o sp h a te group o f ATP was a t t a c k e d by the e x t r a c t enzyme, and th e n n e v e r more t h a n 26 p e r c e n t . There seems to be no b a s i s f o r a ssu m in g t h a t th e ATP-ase a c t i v i t y f o r f e r t i l i z e d eggs i s d i f f e r e n t from t h a t of u n f e r t i l i z e d e g g s. No B -G ly c e ro p h o s p h a ta s e a c t i v i t y was d e m o n s tra te d in any o f th e p r o t e i n s o l u t i o n s , b u t a weak one was n o te d i n th e u n f e r t i l i z e d homogenate a f t e r 20 h o u r s 1 i n c u b a t i o n . I t i s c o n clu d ed t h a t a p h o s p h a ta s e s p e c i f i c f o r sodium b g ly c e r o p h o s p h a te was a b s e n t from a l l p r o t e i n p r e p a r a t i o n s . I t i s a ls o e v id e n t fro m th e s e r e s u l t s t h a t a s p e c i f i c ATP- 60 a se i s p r e s e n t . E l e c t r o n m ic ro g ra p h s of m yosin a n d the M irsky p r o t e i n were made and a re shown f o r th e i n t e r e s t th e y convey. I t i s f e l t t h a t a c r i t i c a l d i s c u s s i o n can n o t be made a t t h i s tim e as to t h e i r s i m i l a r i t y . CHAPTER IV SUM M ARY 1. The p r o t e i n of s e a u r c h i n eggs d e s c r i b e d by M irsky h as been i s o l a t e d * E l e c t r o p h o r e t i c a n a l y s i s i n d i c a t e s a homogeneous p r o d u c t. 2 . Chemical and p h y s i c a l t e s t s have e s t a b l i s h e d th e f a c t t h a t th e m ajo r p o r t i o n i s not a n u c l e o p r o t e i n . A c t i v i t y t e s t s f o r ATP-ase were n e g a t i v e . DRF was d e m o n s tr a te d . 3* Homogenates of f e r t i l i z e d and u n f e r t i l i z e d s e a u r c h i n eggs c o n ta in e d a s p e c i f i c A T P-ase, th e f e r t i l i z e d egg h a v in g th e g r e a t e r a c t i v i t y per mg. o f n i t r o g e n . 4 . A lk a lin e KCl e x t r a c t s o f th e above hom ogenates c o n t a i n e d th e A T P-ase, b u t i n t h i s c a se th e e x t r a c t from th e u n f e r t i l i z e d eggs was m ost a c t i v e p e r mg. o f n i t r o g e n , i n b o t h c a s e s i t was found t h a t i n 0 .0 1 M c o n c e n t r a t i o n s , magnesium i o n i n h i b i t e d and c a lc iu m io n a c t i v a t e d th e ATP-ase p r e s e n t . The i n h i b i t o r y e f f e c t of magnesium was p a r t l y o f f s e t by th e a d d i t i o n o f c a lc iu m . 5 . The e x p e r im e n ta l e v id e n c e d o es not w a r r a n t th e a s s u m p tio n t h a t t h e r e i s more ATP-ase p r e s e n t i n th e s e a u r c h i n egg s h o r t l y a f t e r f e r t i l i z a t i o n . 6 . E l e c t r o n m ic ro g ra p h s w ere made of th e M irsky p r o t e i n from u n f e r t i l i z e d e g g s. BIBLIOGRAPHY A x e lro d , B. 1947. C i t r u s f r u i t p h o s p h a ta s e , j o u r . B i o l . Chem. . 165; 5 7 -7 2 pp. B a i l e y , K. 1939. M yosin and a d e n o s i n e t r i p h o s p h a t a s e . Biochem . J o u r . , 36; 121-139 pp. B anga, I . , and S z e n t- G y o r g y i , A. 1940. S t r u c t u r e p r o t e i n s . S c i e n c e , 92; 514-515 pp. Beams, A. and K ing, R. 1936. S u r v iv a l o f A s c a r is eg g s a f t e r c e n t r i f u g i n g . S c i e n c e . , 8 4 :1 3 8 . B e n s le y , R. and H o e rr, u . 1934. S t u d i e s on c e l l s t r u c t u r e by th e f r e e z i n g - d r y i n g m ethod. V The c h em ical b a s i s o f the o r g a n i z a t i o n of th e c e l l . A n a t. R ec. , 6 0 :2 5 1 -2 6 6 . B e n s le y , R. 1938. P la s m o s in . T hegel and f i b e r fo rm in g , c o n s t i t u e n t o f th e p ro to p la sm o f th e h e p a t ic c e l l . A n a t. R e c . , 72; 351-369 pp. B id d u lp h , C . ; M eyer, R . ; and MeShan, W. 1946 a . A d e n o s i n e t r i p h o s p h a ta s e a c t i v i t y and w e ig h t o f e o rp e a l u t e a d u r i n g r e p r o d u c t i v e cy cle o f th e r a t . p r o c . S o c . E x p t l . B i o l . Med. , 62; 36-38 pp. ______ t 1946 b . A d e n o s in e tr ip h o s p h a ta s e a c t i v i t y of l u t e i n and o v a r i a n t i s s u e s and w e ig h t o f c o rp e a l u t e a d u r i n g th e r e p r o d u c t i v e c y c le o f th e r a t . E n d o c rin o lo g y , 38; 358-367 pp. D is c h e , Z. 1930. M ic k ro ch e m ie , 8 :4 p. D uB o is, K. and P o t t e r , V* 1943. The a s s a y of anim al t i s s u e s f o r r e s p i r a t o r y enzym es. I I I . A d e n o s i n e t r i p h o s p h a ta s e . J o u r . B i o l . Chem. , 150: 185-195 pp. E d s a l l , J . and M ehl, J . 1940. The e f f e c t of d e n a t u r i n g a g e n ts on m yosin. I I . V i s c o s i t y and d o u b le r e f r a c t i o n of flo w . J o u r . B i o l . Chem. 133: 4 0 9 -4 3 0 . E d s a l l , J . 1942. S tre a m in g b i r e f r i n g e n c e a n d i t s r e l a t i o n to p a r t i c l e s i z e and s h a p e . Advances i n c o l l o i d s c i e n c e . 1: 2 69 -3 16. Hew Y ork: I n t e r s c l e n c e . 63 E d s a l l , J • ; Gordon, C .: and M ehl, J . 1944. S t u d i e s on d o u b le r e f r a c t i o n o f flo w . I . An a p p a r a t u s f o r th e s tu d y of dou ble r e f r a c t i o n o f flo w a t h ig h v e l o c i t y g r a d i e n t s . Review o f S c i e n t i f i c i n s t r u m e n t s , 15: 243-252 pp: E n g e l h a r d t , W. and lyubim owa, M. 1939. Myosin and a d e n i s o n e t r i p h o s p h a t a s e . N a t u r e , 144; 668-669 pp. F i s k e , C. and Subbarow, Y. 1925. The c o l o r i m e t r i c d e t e r m i n a t i o n of p h o s p h o ru s . J o u r . B i o l . Chem., 66: 375-400 pp. -------- -------- G r e v i l l e , G. and Lehmann, H. 1943. M agnesium -calcium a n ta g o n ism i n m u sc le . N a t u r e , 152: 81-82 pp. H e ilb r u n n , L. 1937. S t i m u l a t i o n and n u c l e a r breakdown i n th e N e re is e g g . B i o l . B u l l . , 73: 557-564 pp. 1940. The r e a c t i o n o f c a lc iu m on m uscle p r o to p la s m . P h y s . Z o o l. , 13: 88-94 p p. H u tc h e n s, J . ; K e l tc h , A .; K r a h l, M .; and Clowes, G. 1942. S t u d i e s on c e l l m e ta b o lism and c e l l d i v i s i o n . V I. O b s e r v a tio n s of t h e g ly co g en c o n t e n t , c a rb o h y d ra te c o n su m p tio n , l a c t i c a c i d p r o d u c t i o n , and ammonia p r o d u c t i o n of eggs of A rb a c ia p u n c t u l a t a . j o u r . Gen. P h y s i o l . , 25: 717-731. L e r n e r , A. and Barnum, C. 1946. The u l t r a v i o l e t a b s o r p t i o n o f plasm a p r o t e i n s . A rc h . Biochem . , 10: 417-425 pp . L ie b k n e c h t, W. 1939. U ber die a u f s p a l t u n g d e r a d e n y l- p y ro p h o sp h o sa u re d u rc h k n o c h e n p h o s p h a ta s e . B ioehem isehe Z e i t s c h r i f t , 303: 96-100 pp. L in d b e rg , 0. 1946. On th e o c c u rre n c e of p r o p a n e d io l p h o sp h a te and i t s e f f e c t on th e c a rb o h y d ra te m e ta b o lism i n a n im al t i s s u e s . A r k iv . f o r Kemi, M i n e r a l o g i , och G e o lo g i, Band 23R. Mac Leod, J . and Summerson, W. 1946. The p h o s p h a ta s e a c t i v i ty o f human sp e rm a ta z o a . J o u r . B i o l. Chem., 165; 533- 539 pp. Mehl, J . and S e x to n , E. 1943. E f f e c t of s a l t and s u b s t r a t e c o n c e n t r a t i o n upon a c t i v i t y of a d e n o s i n e t r i p h o s p h a t a s e . P r o c . Soc. E x p t l . B i o l . M e d ., 52: 38-40 pp. 64 M ehl, J • 1938, Double r e f r a c t i o n o f flow or p r o t e i n s o l u t i o n s . Cold S p r in g H arb or Sym posia on Q u a n ti- t a t i v e B i o lo g y , VI: '218-227 pp. M irsk y , A. 1936a. p r o t e i n c o a g u l a t i o n as a r e s u l t o f f e r t i l i z a t i o n . S c i e n c e , 84: 333-3334. ___________ , 1936b. The change i n s t a t e o f the p r o t e i n s o f m uscle i n r i g o r . J o u r . Gen. phys i o l . , 19; 571-575 pp. , 1938. p r o t e i n d e n a t u r a t i o n . Cold S p rin g H arbor Sym posia on Q u a n t i t a t i v e B io lo g y , 6: I b 0 - i b 3 pp. , and P o l l i s t e r , A. 1946. C hrom osin, a d e s o x y rib o s e n u c l e o p r o t e i n complex o f th e c e l l n u c l e u s . J o u r . Gen. P h y s i o l . , 30: 117-148. Moog, F. and S t e in b a c h , H. 1945. A d e n y lp y ro p h o sp h a ta se i n c h ic k em bryos. J o u r . c e l l . Comp, p h y s i o l . , 25: 133-144 pp. Moore, A. and M i l l e r , W. 1937. O c cu rre n ce o f b i r e f r i n g e n c e i n th e f e r t i l i z e d egg o f th e s e a u r c h in * p r o c . Soc. F x p t l . B i o l . M e d ., 36: 835-836 pp. P o l l i s t e r , A. and M irsk y , A. 1946. The n u c le o p ro ta m in e o f t r o u t sperm . Jour. Gen. p h y s i o l . , 30: 101-116 pp. S e h e e r, B. and S c h e e r, M. 1946. Some i n t e r r e l a t i o n s o f d ru g and io n a c t i o n s i n th e a r t i f i c i a l a c t i v a t i o n o f m arine e g g s. P h y s . Z o o l . , XX: 15-32 pp. S c h m id t, G. and T h arm hauser, S. 1943. I n t e s t i n a l p h o s p h a ta s e J o u r . B i o l . Chem. , 149: 369-385 pp. S in g h e r , H. and Mei s t e r , A. 1945. The a n e n o s i n e t r i p h o s p h a t a s e a c t i v i t y o f m yosin p r e p a r a t i o n s , j o u r . B i o l . Chem. 159: 491-501 pp. S te in b a c h , H. and Moog, F. 1945. L o c a l i z a t i o n of a d e n y lp y ro p h o s p h a ta s e i n c y to p la s m ic g r a n u l e s , j o u r . C e l l . Comp. P h y s i o l . , 26: 175-183 pp. U m b re it, W .5 B u r r i s , R . ; and S t a u f f e r , V. 1945. M anom etric te c h n iq u e s and r e l a t e d m ethods f o r th e s t u d y o f t i s s u e m e ta b o lism . M in n e a p o lis : B u rg e ss p u b l i s h i n g company. 65 V ie s , F . 1926. I n t e r n a l pH o f t h e s e a u r c h i n e g g . Comp. Rend. Soc. B i o l * ( P a r i s ) , 94; 4 6 9 -4 7 1 . ______ .» and Gex, M * 1928. Sur l e s p e c t r e u l t r a v i o l e t de l r o e u f d ’ o u r s i n ( P a r a c e n t r o t u s l i v i d u s L s ) e t de s e s c o n s t i t u a n t s . A rch, de p h y siq u e B io lo g i q u e , b: 255-286 pp. W eber, H* H*, and Meyer, K. 1933. C o l l o i d a l b e h a v io r o f m uscle p r o t e i n s , V. Q u a n t i t a t i v e r e l a t i o n s h i p betw een m uscle p r o t e i n s and i t s s i g n i f i c a n c e f o r th e s t r u c t u r e of s t r i a t e d r a b b i t m u sc le . Bioehem. z . , 266: 137-152 pp.

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Creator Connors, William Matthew (author)

Core Title Properties of the myosin-like proteins of sea urchin eggs

Degree Master of Science

Degree Program Biochemistry and Nutrition

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag chemistry, biochemistry,OAI-PMH Harvest

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Advisor [illegible] (committee chair), Bartholomew, James W. (committee member), Mehl, John W. (committee member)

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