Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Creatine kinase in Xenopus laevis development : changes in enzyme activity and isozyme patterns Haswell, Polly 1976

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

Item Metadata

Download

Media
831-UBC_1977_A6_7 H38.pdf [ 6.33MB ]
Metadata
JSON: 831-1.0094011.json
JSON-LD: 831-1.0094011-ld.json
RDF/XML (Pretty): 831-1.0094011-rdf.xml
RDF/JSON: 831-1.0094011-rdf.json
Turtle: 831-1.0094011-turtle.txt
N-Triples: 831-1.0094011-rdf-ntriples.txt
Original Record: 831-1.0094011-source.json
Full Text
831-1.0094011-fulltext.txt
Citation
831-1.0094011.ris

Full Text

CREATINE KINASE IN XENOPUS LAEVIS DEVELOPMENT: CHANGES IN ENZYME ACTIVITY AND ISOZYME PATTERNS by POLLY HASWELL . S. C a l i f o r n i a S t a t e U n i v e r s i t y , Long Beach 19 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF - THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the department o f Zoology We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d The U n i v e r s i t y o f B r i t i s h Columbia November, 1976 (7) Polly Haswell, 1976 In presenting th is thes is in p a r t i a l fu l f i lment o f the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary shal l make it f ree ly ava i l ab le for reference and study. I fur ther agree that permission for extensive copying of th is thes is for scho la r ly purposes may be granted by the Head of my Department or by h is representat ives . It is understood that copying or pub l ica t ion of th is thes is fo r f inanc ia l gain sha l l not be allowed without my wri t ten permission. Department of The Univers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 D a t e <mJLm*Auj 4 1^7^ i ABSTRACT The early developmental changes of creatine kinase have been described i n Xenopus l a e v i s , the South A f r i c a n clawed frog.. The s p e c i f i c a c t i v i t y was low during early dev-elopment and increased about 5-fold between stages 4-2 and 48. Isozyme I i s present from the u n f e r t i l i z e d egg to stage 42 (free-swimming l a r v a ) , when isozyme II i s f i r s t detected. Isozyme III f i r s t appeared i n post-metamorphic frogs. Iso-zymes II and III have been l o c a l i z e d i n the muscle tissue, but isozyme II i s absent i n adult muscle tissue suggesting i t may be a transient, embryonic form of CK. An attempt was made to induce the precocious appearance of isozyme III by tre a t i n g premetamorphic tadpoles with triiodothyronine. i i TABLE OF CONTENTS Page INTRODUCTION v • 1 MATERIALS AND METHODS P r e p a r a t i o n o f Embryos and E x t r a c t s f o r Assa y s • • 9 P r e p a r a t i o n o f A d u l t T i s s u e s and Embryos f o r Agarose G e l E l e c t r o p h o r e s i s 10 Assay o f C r e a t i n e K i n a s e . . l u Assay o f C i t r a t e S y n t h e t a s e 13 Assay o f L a c t a t e Dehydrogenase 13 P r o t e i n D e t e r m i n a t i o n s . 13 Agarose G e l E l e c t r o p h o r e s i s 14 I n d u c t i o n o f Metamorphosis 15 RESULTS S p e c i f i c A c t i v i t y o f C r e a t i n e K i n a s e . . . . . . . . . . . 16 C o n t r o l o f M i t o c h o n d r i a l Breakage 21 A d u l t Isozyme P a t t e r n s 22 Ontogeny o f C r e a t i n e K i n a s e Isozymes........... 22 I n d u c t i o n o f Metamorphosis 27 DISCUSSION . : 4 i SUMMARY 5 4 REFERENCES 5 5 i i i L IST OF FIGURES Page F i g u r e 1. Graph o f S p e c i f i c A c t i v i t y v s . Stage o f Development 17 F i g u r e 2. Graph o f S p e c i f i c A c t i v i t y v s . Stage o f Development—Two I n d i v i d u a l E x p e r i m e n t s • 19 F i g u r e 3. C r e a t i n e K i n a s e Isozyme P a t t e r n s o f A d u l t Organs o f Xenopus l a e v i s 23 F i g u r e 4. C r e a t i n e K i n a s e Isozyme P a t t e r n s o f E a r l y Embryos, U n f e r t i l i z e d Egg t o Stage 42 2 5 F i g u r e 5. C r e a t i n e K i n a s e Isozyme P a t t e r n s o f Xenopus l a e v i s L a r v a e Showing M i c r o h e t e r g e n e i t y 2 8 F i g u r e 6. C r e a t i n e K i n a s e Isozyme P a t t e r n s o f Xenopus l a e v i s a t L a t e r Developmental Stages 3 0 F i g u r e 7. C r e a t i n e K i n a s e Isozyme P a t t e r n s o f Regions o f Stage 5 8 T a d p o l e s 32 F i g u r e 8. C r e a t i n e K i n a s e Isozyme P a t t e r n s o f Stage 54 and 5 8 Tadpoles T r e a t e d w i t h T r i i o d o t h y r o n i n e (T^) 35 F i g u r e 9. C r e a t i n e K i n a s e Isozyme P a t t e r n s o f Stage 54 Tadpoles T r e a t e d w i t h T 3 f o r One Week 37 F i g u r e 10. C r e a t i n e K i n a s e Isozyme P a t t e r n s o f Stage 54 Tad p o l e s T r e a t e d w i t h T 3 f o r Two Weeks 39 1 INTRODUCTION C e l l d i f f e r e n t i a t i o n involves, at le a s t i n part, d i f f e r e n t -i a l gene a c t i v i t y and control of synthesis and degradation o f s p e c i f i c proteins i n d i f f e r e n t regions of the embryo and at d i f f e r e n t times during development. One approach to studying genetic and epigenetic control during development i s to sel e c t a p a r t i c u l a r enzyme and analyse i t s properties, i t s l o c a l i z a t i o n , and the factors that regulate i t s synthesis during development. Changes i n isozyme patterns, i n a number of cases, have been shown to represent changes of at l e a s t two gene products. Iso-zyme patterns change during development i n an i n d i v i d u a l i s t i c manner i n d i f f e r e n t regions and at d i f f e r e n t times; however, very l i t t l e i s known about whether these changes are controlled at the t r a n s c r i p t i o n a l , t r a n s l a t i o n a l , or p o s t - t r a n s l a t i o n a l l e v e l . In addition, isozyme patterns r e f l e c t the state o f d i f f e r e n t i a t i o n of s p e c i f i c tissues and can be correlated with p h y s i o l o g i c a l and morphological changes i n the developing organism. Isozyme patterns, therefore, can be u t i l i z e d as bio-chemical correlates of s p e c i f i c developmental events and are a useful t o o l with which to study determination and d i f f e r e n t i a -t i o n of s p e c i f i c c e l l types. Markert and Moller (1959) suggested the use of the term isozymes to describe enzymes which catalyse the same reaction but which e x i s t i n multiple molecular forms i n the same organism, tissue or c e l l . Isozymes, or isoenzymes, are usually separated. 2 by e lec t rophores i s and i d e n t i f i e d by coupl ing the s p e c i f i c enzyme a c t i v i t y to the depos i t ion of an inso lub le formazan dye,. Lactate dehydrogenase (LDH) i s the archetypal example of an isozyme system.: I t was with t h i s enzyme that Markert and Ursprung (1962) c l e a r l y demonstrated the ontogeny of isozyme-patterns during the development of the mouse. Complex patterns c h a r a c t e r i s t i c of adult t i s sue types usua l ly a r i se from a common embryonic precursor pa t tern . Subsequent research has., shown that isozyme patterns a r i se from the random assoc ia t ion of d i s s i m i l a r polypeptide chains to form multimeric enzyme molecules (Eppenberger, 1975) . Changes i n isozyme patterns presumably r e f l e c t changes i n c e l l u l a r metabolism. The study of the cont ro l of isozyme patterns during embryo-genesis presents a f a sc ina t ing problem, s ince a number of steps may intervene from the t r a n s c r i p t i o n of the gene to the forma-t i o n of a funct iona l enzyme molecule. Changes i n isozyme patt-erns are probably due l a r g e l y to d i f f e r e n t i a l gene regu la t ion , but epigenet ic regulatory mechanisms must also be considered (Whitt, 1975). A s p e c i f i c mRNA may have i t s access to the cytoplasm regulated, and once i t does reach the cytoplasm, t r a n s l a t i o n may be regulated. Assembly of polypeptide subunits may be c o n t r o l l e d ; the d i f f e r e n t i a l degradation and/or synthesis of subunits may be responsible for the observed c e l l - s p e c i f i c pat terns . Only ac t ive enzyme molecules w i l l be detected by the methods used to resolve isozyme pat terns , and a number of factors a f fec t enzyme a c t i v i t y without changing t h e i r amounts. 3 Enzyme a c t i v i t y may be i n f l u e n c e d by i n h i b i t o r s o r o t h e r a l l o -s t e r i c m o d i f i c a t i o n s . D u r i n g development and d i f f e r e n t i a t i o n a f u n c t i o n a l enzyme m o l e c u l e may r e s u l t from th e a c t i v a t i o n o f an i n a c t i v e p r e c u r s o r (de V e l l i s , 1970). C o m p a r t m e n t a l i z a t i o n o f an enzyme may a l s o be i n v o l v e d i n r e g u l a t i n g i t s a c t i v i t y . The ontogeny o f isozyme p a t t e r n s has been d e s c r i b e d f o r numerous enzymes i n many o r g a n i s m s , bu t v e r y l i t t l e i n f o r m a t i o n i s a v a i l a b l e c o n c e r n i n g the i n t r a c e l l u l a r c o n t r o l o f t h e s e p a t t e r n s d u r i n g a n i m a l development. Lebherz (1975a) a s c r i b e d t h e e s t a b l i s h m e n t o f a l d o l a s e isozyme p a t t e r n s t o d i f f e r e n t i a l r a t e s o f s y n t h e s i s o f s u b u n i t s , whereas F r i t z e t a l . (1975) have i n t e r p r e t e d t h e appearance o f LDH p a t t e r n s t o be due t o d i f f e r e n t i a l r a t e s o f s y n t h e s i s as w e l l as d i f f e r e n t i a l r a t e s o f d e g r a d a t i o n . D u r i n g t h e development o f c h i c k e n s k e l e t a l m u s c l e , t h e r e i s a t r a n s i t i o n o f a l d o l a s e .from t e t r a m e r s con-t a i n i n g o n l y C s u b u n i t s t o t e t r a m e r s c o n t a i n i n g o n l y A s u b u n i t s (Lebherz and R u t t e r , 1969), w i t h h e t e r o m e r i c a l d o l a s e t e t r a m e r s b e i n g produced d u r i n g the t r a n s i t i o n . The q u a r t e r n a r y s t r u c t u r e o f a l d o l a s e isozymes i s s t a b l e ( L e b h e r z , 1975b) t h e r e f o r e such t r a n s i t i o n s o c c u r w i t h i n i n d i v i d u a l c e l l s . The r e g u l a t i o n o f a l d o l a s e s u b u n i t c o n c e n t r a t i o n d u r i n g development was i n v e s t i -g a t e d by Lebherz (1975a), and the i n c r e a s e d a c c u m u l a t i o n o f a l d o l a s e A4 w^ s^ a t t r i b u t e d t o an i n c r e a s e d r a t e o f s y n t h e s i s o f A s u b u n i t s . The e v i d e n c e i s i n c o n c l u s i v e t h a t t h e a c c u m u l a t i o n o f t h e A 4 enzyme i s due t o an i n c r e a s e i n t h e r a t e o f s y n t h e s i s o f t h e A s u b u n i t . R a d i o a c t i v e amino a c i d s were a d m i n i s t e r e d 4 i n t r a p e r i t o n e a l l y t o young c h i c k s , b u t the p o o l s i z e s o f the amino a c i d s were n o t measured i n t h e t i s s u e s t u d i e d . I n o r d e r t o d e t e r m i n e th e r a t e o f m a c r o m o l e c u l a r s y n t h e s i s i t i s n e c e s s -a r y t o know what i s happening t o the p o o l s i z e ( M i t c h i s o n , 1971) . I t i s e v i d e n t t h a t t h e r e i s an i n c r e a s e d a c c u m u l a t i o n o f A sub-u n i t s , r e l a t i v e t o the C s u b u n i t s , b u t i t has not been e s t a b -l i s h e d t h a t t h e a c c u m u l a t i o n i s due t o an i n c r e a s e d r a t e o f s y n t h e s i s o f A s u b u n i t s r a t h e r t h a n an i n c r e a s e d r a t e o f d e g r a d a t i o n o f C s u b u n i t s . F r i t z e t al_. (1975) s t u d i e d t h e i n t r a c e l l u l a r t u r n o v e r r a t e o f isozymes o f LDH d u r i n g t h e p o s t -n a t a l development o f r a t h e a r t , l i v e r , and s k e l e t a l muscle. These workers found t h a t b o t h the r a t e o f s y n t h e s i s as w e l l as t h e r a t e o f d e g r a d a t i o n may e s t a b l i s h t h e l e v e l s o f isozymes p r e s e n t i n a s p e c i f i c t i s s u e t y p e . Assuming t h a t t h e b a s i c c o n t r o l mechanism g e n e r a t i n g isozyme p a t t e r n s i s the r e g u l a t i o n o f the p r o d u c t i o n o f p o l y p e p t i d e u n i t s i n each c e l l , t h e n i t would be i n t e r e s t i n g t o d e t e r m i n e i f the r a t e o f s y n t h e s i s i s d e t e r m i n e d a t t h e t r a n s c r i p t i o n a l and/or t r a n s l a t i o n a l l e v e l o f p r o t e i n s y n t h e s i s . I t i s d i f f i c u l t t o e s t a b l i s h the mechanisms i n v o l v e d i n i n t r a c e l l u l a r c o n t r o l o f isozyme p a t t e r n s , and most s t u d i e s con-c e r n i n g the ontogeny o f isozyme p a t t e r n s n e c e s s a r i l y have been o f a d e s c r i p t i v e n a t u r e . I n t h i s s t u d y I examined changes i n c r e a t i n e k i n a s e enzyme a c t i v i t y and the t e m p o r a l and s p a t i a l d i s t r i b u t i o n o f isozyme p a t t e r n s i n a d u l t and d e v e l o p i n g Xenopus l a e v i s ( h e r e a f t e r r e f e r r e d t o as Xenopus). C r e a t i n e k i n a s e (adenosine 5 ' - t r i p h o s -5 phate-creatine phosphotransferase, EC 2.7.3.2) catalyses the following reaction: Creatine Phosphate + ADP ATP + Creatine Creatine kinase (CK) catalyzes a reaction where a l l steps are i n rapid-equilibrium (Morrison and James, 1965), and enzyme a c t i v i t y i s not i n h i b i t e d by high ATP concentrations (Watts, 1973). The energy for use i n c o n t r a c t i l e systems i s derived from ATP, and CK serves to regenerate ATP from ADP and creatine phosphate. CK i s c h a r a c t e r i s t i c of muscle tissue and represents 10-20% of the soluble sarcoplasmic protein i n s k e l e t a l muscle (Watts, 1973). In mammals and chickens, creatine kinase occurs as three forms distinguished by t h e i r electrophoretic mobility (Eppenberg-er et a l . , 1964), a muscle (MM), a hybrid (MB), and a brain-type (BB) (Dawson e t a l . , 1965). These CK's are dimers of approxi-mately 84,000 daltons, formed by the association of 2 types of subunits, M and B (Dawson et a l . , 1965, 1967; Eppenberger et a l . , 1967). The 2 subunits are products of 2 genes as determined by peptide mapping, amino acid analysis, and immunological behavior (Eppenberger et al_. , 1967) . The ontogeny of CK has been described for rats and chickens (Eppenberger et a l . , 1964), the green sunfish (Champion and Whitt, 1976) and Rana temporaria (Lyzlova and Guncheva, 1973). During development of rats and chickens the brain-type isozyme i s present at the e a r l i e s t stages studied, and there i s a gradual progression to the muscle-type as development proceeds. The green sunfish presents quite a d i f f e r e n t s i t u a t i o n i n which a slow-migrating form appears f i r s t , followed by brain and muscle 6 i s ozymes. I n Rana t e m p o r a r i a , the isozyme p a t t e r n s i n t h e c a u d a l and c e p h a l i c r e g i o n s do not change t h r o u g h o u t th e p e r i o d s t u d i e d , from 8 days t h r o u g h 30 days p o s t - h a t c h i n g . The b r a i n isozyme i s p r e s e n t i n the c e p h a l i c r e g i o n and two d i f f e r e n t isozymes a r e found i n t h e c a u d a l r e g i o n s . The s u g g e s t e d p h y s i o l o g i c a l f u n c t i o n o f m u l t i p l e m o l e c u l a r forms o f enzymes i s t o meet the s p e c i f i c m e t a b o l i c r e q u i r e m e n t s o f p a r t i c u l a r c e l l o r t i s s u e t y p e s (Masters and Holmes, 1972). There i s c o n f l i c t i n g e v i d e n c e c o n c e r n i n g t h e p h y s i o l o g i c a l s i g -n i f i c a n c e o f CK i sozymes. The p u r i f i e d MM and BB isozymes from r a b b i t and c h i c k e n s k e l e t a l muscle and b r a i n were r e p o r t e d t o d i f f e r s u b s t a n t i a l l y i n t h e i r c a t a l y t i c c h a r a c t e r i s t i c s (Eppen-b e r g e r e t a l . , 1967). The b r a i n enzyme had l o w e r Km's f o r ADP, c r e a t i n e phosphate (CP) and c r e a t i n e t h a n d i d t h e muscle enzymes. The h y b r i d e x h i b i t e d i n t e r m e d i a t e c a t a l y t i c p r o p e r t i e s (Dawson e t a l . , 1965). However, a c c o r d i n g t o Eppenberger e t a l . (1975), the p h y s i o l o g i c a l s i g n i f i c a n c e o f t h e t r a n s i t i o n was not c l e a r , and they s u g g e s t e d t h a t the i n t r a c e l l u l a r l o c a l i z a t i o n o f t h e MM isozyme was s i g n i f i c a n t r a t h e r than any c a t a l y t i c d i f f e r e n c e s . I n c h i c k e n s k e l e t a l muscle th e MM isozyme was bound t o t h e M-r e g i o n o f t h e m y o f i b r i l , and the BB form o f CK o c c u r r e d i n c h i c k -en h e a r t and was l o c a l i z e d on the Z-band o f t h e c a r d i a c muscle m y o f i b r i l s . The s u g g e s t e d f u n c t i o n would be t o a l l o w f o r more e f f i c i e n t muscle f u n c t i o n because o f the p r o x i m i t y o f the ATP r e g e n e r a t i n g and ATP h y d r o l y s i n g s i t e s . 7 In a d d i t i o n t o l o c a l i z a t i o n o f CK on t h e sarcomere, CK has a l s o been found i n h e a r t m i t o c h o n d r i a (Jacobus and L e h n i n g e r , 1973; S t o r e y , 1974) ; however, i t s r e l a t i o n s h i p t o t h e t h r e e p r i n c i p a l isozymes i s u n c l e a r (Turner, 1975) . Isozyme changes can o f t e n be c o r r e l a t e d w i t h s p e c i f i c de-v e l o p m e n t a l e v e n t s . The t r a n s i t i o n o f isozyme p a t t e r n s o f c r e a t i n e k i n a s e o c c u r s d u r i n g the i n v i t r o d i f f e r e n t i a t i o n o f c h i c k and r a t m y o b l a s t s t o form m u l t i n u c l e a t e d c r o s s - s t r i a t e d , c o n t r a c t i l e myotubes (Turner e t a l . , 1974; S h a i n b e r g e t a l . , 1971; K e l l e r and Nameroff, 1974; T u r n e r , 1975). T u r n e r e t a l . (1976) have e s t a b l i s h e d t h a t CK i s i n d i c a t i v e o f t e r m i n a l s k e l e -t a l muscle c e l l d i f f e r e n t i a t i o n i n v i t r o . I n t h i s s t u d y I examined the t e m p o r a l changes o f CK a c t i v i t y and the t e m p o r a l and s p a t i a l changes o f isozyme p a t t e r n s d u r i n g Xenopus embryogenesis. I n i t i a l l y t h e s e s t u d i e s were u n d e r t a k e n t o d e t e r m i n e t h e o n s e t o f myogenesis i n Xenopus, and CK was chosen as a b i o c h e m i c a l marker o f the s t a t e o f d i f f e r e n t i a t i o n o f muscle t i s s u e . As the d a t a p r e s e n t e d i n t h i s t h e s i s have shown, CK isozyme t r a n s i t i o n s from t h e embryonic p a t t e r n t o a p a t t e r n c h a r a c t e r i s t i c o f a d u l t s k e l e t a l muscle t i s s u e o c c u r r e d d u r i n g metamorphosis, w e l l a f t e r the i n i t i a t i o n o f myogenesis. T h e r e f o r e , an attempt was made t o induce the p r e c o c i o u s appear-ance o f t h e a d u l t n u i s c l e - s p e e i l'ic CK isozyme by t r e a t i i u j pre-metamorphic t a d p o l e s w i t h exogenous t h y r o i d hormone. As no p r e v i o u s work on t h e ontogeny o f CK i n t h i s anuran has been p u b l i s h e d t h e p r e s e n t work w i l l a t l e a s t add t o t h e b i o c h e m i c a l 8 c h a r a c t e r i z a t i o n o f an o r g a n i s m w e l l s u i t e d f o r t h e study o f m o l e c u l a r d e v e l o p m e n t a l b i o l o g y (Deuchar, 1975). MATERIALS AND METHODS P r e p a r a t i o n o f Embryos and E x t r a c t s f o r Enzyme A s s a y s : O v u l a t i o n and mating were i n d u c e d a c c o r d i n g t o the p r o c e d -u r e s o f Gurdon (1967). The a n i m a l s were a l l o w e d t o mate o v e r -n i g h t i n Brown's s o l u t i o n (Brown and C a s t o n , 1962). The j e l l y c o a t s o f eggs and embryos were removed t h e f o l l o w i n g morning by t r e a t m e n t w i t h 2% c y s t e i n e , pH 7.8 f o r a p p r o x i m a t e l y two m i n u t e s . The d e j e l l i e d embryos were r e a r e d i n . Brown's s o l u t i o n a t room t e m p e r a t u r e . Developmental s t a g e s were d e t e r m i n e d a c c o r d i n g t o t h e normal t a b l e s o f Xenopus l a e v i s (Nieuwkoop and F a b e r , 1967). The embryos were c o l l e c t e d a t v a r i o u s s t a g e s and homogenized i n 2-10 volumes o f 0.01 M T r i s , pH 7.5, c o n t a i n i n g 5 mM M g + +. (The volume o f e x t r a c t i o n b u f f e r was v a r i e d , a c c o r d i n g t o t h e s t a g e o f development, t o g i v e the d i l u t i o n n e c e s s a r y t o measure the o p t i c a l d e n s i t y changes w i t h i n the l i m i t s o f the enzyme assay.) The homogenates were c e n t r i f u g e d a t 10,000 rpm i n an SS-34 r o t o r o f a S o r v a l l RC2-B r e f r i g e r a t e d c e n t r i f u g e f o r 15 m i n u t e s , t h e s u p e r n a t a n t removed, and t h e p e l l e t resuspended i n the volume o f b u f f e r used f o r the o r i g i n a l e x t r a c t i o n . A l i q u o t s o f e i t h e r t h e s u p e r n a t a n t o r the resuspended p e l l e t were assayed i m m e d i a t e l y f o r enzyme a c t i v i t y , the remainder then f r o z e n and s t o r e d a t -70°C u n t i l p r o t e i n d e t e r m i n a t i o n s were performed. The p r o t e i n r e m a i n i n g i n the s u p e r n a t a n t f r a c t i o n w i l l be r e -f e r r e d t o as e x t r a c t a b l e p r o t e i n . V 10 P r e p a r a t i o n o f A d u l t T i s s u e s and Embryos f o r Agarose G e l  E l e c t r o p h o r e s i s : A d u l t f r o g s were k i l l e d by f r e e z i n g , they were t h e n thawed, the t i s s u e s removed and washed i n i c e c o l d 0.01 M T r i s , pH 7.5 c o n t a i n i n g 5 mM M g + + and 20 mM 2 - M e r c a p t o e t h a n o l ( S t o r e y , 1974). The t i s s u e s were minced w i t h s c i s s o r s , homogenized i n 10 volumes (w/v) o f i c e c o l d b u f f e r and c e n t r i f u g e d a t 10,000 rpm f o r 15 minutes t o c l a r i f y the e x t r a c t . One t o 1.5 p i samples were used f o r e l e c t r o p h o r e s i s . Embryos and t a d p o l e s were homogenized i n a minimum amount o f b u f f e r and then t r e a t e d i n the same manner as t h e a d u l t t i s s u e s . Assay o f C r e a t i n e K i n a s e : C r e a t i n e k i n a s e was assayed a c c o r d i n g t o S t o r e y (1974), w i t h minor m o d i f i c a t i o n s . A l l enzyme a c t i v i t i e s were measured u s i n g a G i l f o r d Model 2400 R e c o r d i n g S p e c t r o p h o t o m e t e r . Sub-s t r a t e s , c o f a c t o r s (except g l u c o s e and Mg + +) and enzymes were p u r c h a s e d from Sigma Chemical Company, S t . L o u i s , Mo. The i n c u b a t i o n s c o n t a i n e d 20 mmoles I m i d a z o l e , pH 6.7, 4 mmoles g l u c o s e , 4 mmoles magnesium a c e t a t e , 1 mmole adenosine d i p h o s p h a t e (ADP), 10 mmoles adenosine monophosphate (AMP), 2 mmoles n i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e phosphate (NADP), 0.005 mis g l u c o s e - 6 - p h o s p h a t e dehydrogenase (G7PDH) 400 u n i t s / n i l , 0.07 nils h e x o k i n a s e s o l u t i o n , 5 nunolos c r e a t i n e phosphate (CP), and 10-75 u l crude enzyme e x t r a c t i n a f i n a l volume o f 1 ml. The h e x o k i n a s e s o l u t i o n was m o d i f i e d from Dawson and Eppenberger (1970) , and c o n s i s t e d o f 12 mg h e x o k i n a s e (26 u n i t s / mg), 1.9 mis I m i d a z o l e b u f f e r , and 0.1 mis 1% b o v i n e serum a l b u m i n . T h i s s o l u t i o n was s t a b l e f o r a b o u t one week i f s t o r e d i n t h e r e f r i g e r a t o r . A l l a s s a y components e x c e p t c r e a t i n e p h o s p h a t e were i n c u -b a t e d a t room t e m p e r a t u r e (20-22 C) f o r 1-2 m i n u t e s , t h e n - C P was a d d e d t o s t a r t t h e r e a c t i o n . CP was u t i l i z e d t o s t a r t t h e r e a c t i o n b e c a u s e a t some d e v e l o p m e n t a l s t a g e s t h e c r u d e embryo e x t r a c t s c o n t a i n e d a c o n s i d e r a b l e amount o f endogenous s u b s t r a t e c ompared t o t h e amount o f a d d e d CP. The i n c r e a s e i n NADPH p r o -d u c t i o n was r e c o r d e d a t 340 nm, a n d t h e r a t e o f ATP p r o d u c e d was c a l c u l a t e d f r o m t h e l i n e a r p o r t i o n o f t h e c u r v e . Enzyme a c t i v i t i e s a r e e x p r e s s e d a s t h e amount o f p r o d u c t f o r m e d p e r m i n u t e u n d e r t h e a s s a y c o n d i t i o n s e m p l o y e d . F o r e v e r y mole o f ATP p r o d u c e d , 1 mole o f NADP was r e d u c e d . The r e a c t i o n s e q u e n c e o f t h e a s s a y i s as f o l l o w s : CK CP + ADP 1> ATP + C r e a t i n e G l u c o s e + ATP H f , X O k . i n ^ e G6P + ADP (1) G6PDH . G6P P» 6PG NADP —& NADPH C o n t r o l s w i t h no CP were a s s a y e d and any b a c k g r o u n d a c t i v i t y was s u b t r a c t e d . The b a c k g r o u n d was p r e s u m a b l y m y o k i n a s e w h i c h c a t a -l y s e s t h e f o l l o w i n g r e a c t i o n : ' M y o k i n a s e AMP + ATP ^ » 2 ADP a n d t h u s c a n a l s o g e n e r a t e ATP. T h i s r e a c t i o n was r e d u c e d by t h e a d d i t i o n o f 10 mmoles AMP t o t h e r e a c t i o n m i x t u r e . 12 The change i n o p t i c a l d e n s i t y p e r minute becomes l i n e a r a f t e r a s h o r t l a g o f about 15-30 seconds. T h i s l a g p e r i o d ob-s e r v e d b e f o r e the appearance o f p r o d u c t becomes l i n e a r i s t y p i c a l o f c o u p l e d enzyme a s s a y s ( S e g e l , 1975). A c c o r d i n g t o S e g e l , t h e r a t e o f p r o d u c t (P) f o r m a t i o n i s p r o p o r t i o n a l t o enzyme c o n c e n t r a t i o n ([E]) under c e r t a i n c o n d i t i o n s . When two c o u p l i n g enzymes a r e u t i l i z e d i n t h e r e a c t i o n , t h e sequence i s : As l o n g as A i s p r e s e n t i n e x c e s s , A> Km f o r E^, and a l l sub-s t r a t e s and c o f a c t o r s a r e s a t u r a t i n g , t h e n as B i s formed i t w i l l be u t i l i z e d by the ex c e s s E 2 , and S i s r a p i d l y c a t a l y s e d t o P f o r m a t i o n . These c o n d i t i o n s have been met i n t h e CK a s s a y , and t h e analagous r e a c t i o n sequence i s : CP ~CK*ATP * G6P - ^ T ^ NADPH (2) (Re f e r t o e q u a t i o n 1 f o r the complete r e a c t i o n . ) The c o n c e n t r a -t i o n o f CP used i n the as s a y s i s much g r e a t e r than t h e Km f o r CK. HK and G6PDH are p r e s e n t i n ex c e s s and the r e a c t i o n s a re dependent o n l y on the r a t e o f f o r m a t i o n o f ATP and G6P, r e s p e c -t i v e l y . A l l o t h e r s u b s t r a t e s and c o f a c t o r s are s a t u r a t i n g . W h i l e CP i s d i s a p p e a r i n g l i n e a r l y from z e r o t i m e , t h e appearance o f NADPH i s l i n e a r a f t e r a s h o r t l a g . When the l i n e a r p o r t i o n o f the cur v e i s used f o r c a l c u l a t i o n o f NADPH, t h e r a t e i s de-pendent o n i y on CK c o n c e n t r a t i o n . < At l e a s t f o u r d e t e r m i n a t i o n s were made f o r each embryo homogenate, and a l i n e a r r e l a t i o n s h i p between enzyme c o n c e n t r a -/ 13 t i o n and change i n o p t i c a l d e n s i t y was o b s e r v e d a t a l l s t a g e s s t u d i e d . An absorbance change from 0.03 t o 0.3 0. D. u n i t s / minute i s w i t h i n the l i m i t s o f t h i s assay p r o c e d u r e . Assay o f C i t r a t e S y n t h e t a s e : C i t r a t e s y n t h e t a s e was a s s a y e d as a b i o c h e m i c a l i n d i c a t o r f o r m i t o c h o n d r i a l breakage a c c o r d i n g t o S r e r e (1969). The i n -c u b a t i o n s c o n t a i n e d 0.5 mmoles a c e t y l coenzyme A, 2.5 mmoles 5 , 5 ' - d i t h i o b i s - ( 2 - n i t r o b e n z o i c a c i d ) , 50 mM T r i s - H C l , pH 8.5, 0.5 mmoles o x a l o a c e t a t e , 10-50 u l crude enzyme e x t r a c t i n a f i n a l volume o f 1 ml. Samples were i n c u b a t e d f o r 1-2 minutes a t room t e m p e r a t u r e as o t h e r enzymes i n the crude e x t r a c t r educed a c e t y l coenzyme A. O x a l o a c e t a t e was u t i l i z e d t o s t a r t t h e r e a c t i o n , and t h e i n c r e a s e i n absorbance o f 5 , 5 ' - d i t h i o b i s -( 2 - n i t r o b e n z o i c a c i d ) was r e c o r d e d a t 412 nm. Assay o f L a c t a t e Dehydrogenase: L a c t a t e dehydrogenase was a s s a y e d t o d e t e r m i n e i f t h e a p p a r e n t i n c r e a s e i n " m i t o c h o n d r i a l " CK a c t i v i t y d u r i n g d e v e l o p -ment was an a r t e f a c t o f the h o m o g e n i z a t i o n p r o c e d u r e (see r e s u l t s ) . The r e a c t i o n m i x t u r e c o n t a i n e d 10 mM g l y c i n e , pH 10.0, 0.1 mmoles n i c o t i n a m i d e adenine d i n u c l e o t i d e (reduced f o r m ) , 10 mmoles p y r u v a t e , and 10-50 y l crude e x t r a c t i n a f i n a l volume o f 1 ml. The r e a c t i o n was i n i t i a t e d by the a d d i t i o n o f p y r u v a t e , and t h e d e c r e a s e i n absorbance o f NADH was r e c o r d e d a t 34 0 nm. P r o t e i n D e t e r m i n a t i o n s : P r o t e i n was e x t r a c t e d a c c o r d i n g t o the method o f Benbow e t a l . (1975), and p r o t e i n c o n t e n t d e t e r m i n e d by the method o f 14 Lowry e t a l . (1951). A l i q u o t s o f embryo e x t r a c t s (0.2 mis) from w h i c h enzyme a c t i v i t y had been d e t e r m i n e d were p r e c i p i t a t -ed w i t h 0.5 mis o f i c e c o l d 25% t r i c h l o r o a c e t i c a c i d (TCA), and a l l o w e d t o s t a n d on i c e f o r . 1 0 m i n u t e s . Samples were c e n t r i -fuged f o r 10 minutes a t 4000 rpm, washed w i t h 5% TCA, r e c e n t r i -f u g ed, t h e n d i s s o l v e d i n 0.2 o r 2 mis o f 0.1 N NaOH t o g i v e t h e a p p r o p r i a t e d i l u t i o n f o r the Lowry method o f p r o t e i n d e t e r m i n a -t i o n (Lowry e t a l . , 1951). Agarose G e l E l e c t r o p h o r e s i s : C r e a t i n e k i n a s e isozymes were r e s o l v e d on agarose g e l s (Beckman p r e p a r e d g e l s ) u s i n g a Beckman Model R-100 M i c r o z o n e e l e c t r o p h o r e s i s system and R-120 power s u p p l y . Agarose g e l e l e c t r o p h o r e s i s was performed u s i n g 0.06 M B a r b i t a l b u f f e r , pH 8.6, c o n t a i n i n g 0.6 mis 2-mercaptoethanol and 4 5 grams o f s u c r o s e p e r l i t e r o f b u f f e r . 1-1.5 u l samples were e l e c t r o -p h oresed a t 150 V and 18 mA f o r 2-4 hours a t 4°C. C r e a t i n e k i n a s e e l e c t r o p h o r e t i c v a r i a n t s were r e v e a l e d by p l a c i n g t h e g e l s i n 10 mis o f t h e f o l l o w i n g s o l u t i o n : 0.1-M T r i s , pH 7.5, 2 0 mmoles CP, 0.5 mmoles NADP, 1.5 mmoles ADP, 0.8 mmoles g l u c o s e , 0.8 mmoles magnesium c h l o r i d e , 0.1 mis h e x o k i n a s e s o l u t i o n , 10 y l G6PDH (400 u n i t s / m l ) , 6 mgs n i t r o -b l u e t e t r a z o l i u m , 0.3 mgs phenazine m e t h o s u l f a t e , and 10 mmoles AMP (to i n h i b i t myokinase a c t i v i t y ) . No enzyme a c t i v i t y was d e t e c t e d when c r e a t i n e phosphate was o m i t t e d from, t h e s t a i n i n g medium. G e l s were s t a i n e d i n the dark u n t i l good c o l o r d e v e l o p -ed ( u s u a l l y about 1 hour a t a room t e m p e r a t u r e o f 23-25°C), 15 t h e n d e s t a i n e d i n 5% a c e t i c a c i d , and d r i e d o v e r n i g h t a t room t e m p e r a t u r e . I n d u c t i o n o f Metamorphosis: Stage 54 and 58 t a d p o l e s were t r e a t e d w i t h 3 , 3 ' , 5 - t r i i o d o -t h y r o n i n e (T^) (Sigma) t o i n d u c e metamorphosis (Rugh, 1965). (Metamorphosis was i n d u c e d when a n i m a l s were t r e a t e d w i t h - 8 - 1 0 10 t o 10 M T 3 ' t n e a n a l o g o f L - t h y r o x i n e t o which Xenopus t a d p o l e s were most s e n s i t i v e [ T a t a , 1968 ].) Tadpoles were s t a g e d , weighed and t h e body l e n g t h and t o t a l l e n g t h measured. A n i m a l s were p l a c e d i n 4" f i n g e r bowls c o n t a i n i n g 200 mis o f Brown's s o l u t i o n , one a n i m a l per d i s h , and the i n c u b a t i n g medium and f o o d ( a c t i v e d r y y e a s t ) were changed d a i l y . E x p e r i m e n t a l a n i m a l s r e c e i v e d , i n a d d i t i o n t o the above, T^ i n f i n a l concen-- 8 - 9 . - 9 t r a t i o n s o f 1.5 X 10 M, 7.5 X 10 M, and 1.5 X 10 M. The CK isozyme p a t t e r n s o f the a n i m a l s were t e s t e d a f t e r v a r i o u s i n t e r -v a l s t o check f o r i n d u c t i o n o f the a d u l t s k e l e t a l muscle isozyme. RESULTS S p e c i f i c A c t i v i t y o f C r e a t i n e K i n a s e d u r i n g Development: C r e a t i n e k i n a s e a c t i v i t y , when e x p r e s s e d as micromoles (pmoles) o f ATP produced/mg e x t r a c t a b l e p r o t e i n / m i n u t e , remains r e l a t i v e l y unchanged t h r o u g h o u t e a r l y embryogenesis and i n c r e a s e s between s t a g e 42 and 48 ( F i g u r e 1 ) . S p e c i f i c a c t i v i t y remains a t a s t e a d y s t a t e from t h e i n i t i a l s t a g e s t u d i e d ( u n f e r t i l i z e d eggs) t h r o u g h s t a g e 20/21 ( c o m p l e t i o n o f n e u r a l tube f o r m a t i o n ) . A t s t a g e 26/27 (spontaneous f l e x i n g movements), the mean s p e c i f i c a c t i v i t y d e c r e a s e s about 3 - f o l d , remains low t h r o u g h s t a g e 33/34 ( h e a r t b e a t ) and r e t u r n s t o t h e l e v e l o f a c t i v i t y o b s e r v e d i n e a r l i e r s t a g e s a t s t a g e 42 ( f r e e swimming t a d p o l e ) . The s p e c i f i c a c t i v i t y r i s e s s h a r p l y between s t a g e 42 and s t a g e 4 8 (premetamorphosis). C o n s i d e r a b l e v a r i a t i o n i n s p e c i f i c a c t i v i t y d u r i n g d e v e l o p -ment i s o b s e r v e d (open c i r c l e s , F i g u r e 1 ) . However, when the s p e c i f i c a c t i v i t y o f i n d i v i d u a l s o b t a i n e d from one s e t o f p a r -e n t s i s compared t o t h e s p e c i f i c a c t i v i t y o f a n i m a l s from a d i f f e r e n t s e t o f p a r e n t s , a s i m i l a r d e v e l o p m e n t a l t r e n d i s a p p a r e n t ( F i g u r e 2 ) . For example, th e s p e c i f i c a c t i v i t y o f progeny from one s e t o f p a r e n t s i s 0.08 ymoles ATP/mg e x t r a c t -a b l e p r o t e i n / m i n u t e a t s t a g e 29/30 and r i s e s t o a maximum o f 2.3 ymoles ATP/mg e x t r a c t a b l e p r o t e i n / m i n u t e a t s t a g e 48. The s p e c i f i c a c t i v i t y o f progeny from a n o t h e r s e t o f p a r e n t s i s 0.2 a t s t a g e 29/30 and r i s e s t o 7.8 a t s t a g e 48. A l t h o u g h e x p e r i -17 F i g u r e 1. A c t i v i t y o f c r e a t i n e k i n a s e i n eggs and d e v e l o p i n g embryos o f Xenopus l a e v i s . S p e c i f i c a c t i v i t y r e f e r s t o the micromoles o f ATP produced/mg p r o t e i n / m i n u t e . Developmental s t a g e s were d e t e r m i n e d a c c o r d i n g t o • the normal t a b l e s o f Nieukoop and Faber (1967). C l o s e d c i r c l e s (•) r e p r e s e n t t h e mean v a l u e s o f 2-6 e x p e r i m e n t s . Open c i r c l e s (0) a r e the a c t u a l v a l u e s o b t a i n e d f o r each e x p e r i m e n t . These v a l u e s a r e t h e average o f a t l e a s t f o u r d e t e r m i n a t i o n s o f absorbance change and t h r e e d e t e r m i n a t i o n s o f p r o t e i n c o n c e n t r a -t i o n s a t each d e v e l o p m e n t a l s t a g e . S P E C I Fl C A C T I V I T Y ( O -U O CO O v V o premetamorphosis 81 19 F i g u r e 2. S p e c i f i c a c t i v i t y o f c r e a t i n e k i n a s e o f d e v e l o p i n g embryos o b t a i n e d from two d i f f e r e n t s e t s o f p a r e n t s ( Wk ) r e p r e s e n t s i n d i v i d u a l s from one s e t ; ( O ) r e p r e s e n t s progeny from a d i f f e r e n t m a t i n g . U n i t s o f s p e c i f i c a c t i v i t y and d e v e l o p m e n t a l s t a g e s a r e as d e s c r i b e d f o r F i g u r e 1. S P E C I F I C A C T I V I T Y NJ 0 s CO OZ 21 m e n t a l v a l u e s o b t a i n e d from progeny o f i n d i v i d u a l matings some-ti m e s v a r y by a f a c t o r o f 10, a d e v e l o p m e n t a l t r e n d i s a p p a r e n t : namely, the CK a c t i v i t y o f e a r l y embryos i s low, and the a c t i v i -t y i n c r e a s e s as development p r o c e e d s . C o n t r o l o f M i t o c h o n d r i a l Breakage: As mentioned p r e v i o u s l y , CK has been l o c a l i z e d i n t h e m i t o c h o n d r i a o f h e a r t t i s s u e from t u r t l e s and b eef ( S t o r e y , 1974; Jacobus and L e h n i n g e r , 1973). To d e t e r m i n e i f p a r t o f t h e o b s e r v e d CK a c t i v i t y was m i t o c h o n d r i a l r a t h e r than c y t o -p l a s m i c , r e l e a s e o f m i t o c h o n d r i a l enzymes was m o n i t o r e d • u s i n g c i t r a t e s y n t h e t a s e , an e x c l u s i v e l y m i t o c h o n d r i a l enzyme, as an i n d i c a t o r o f breakage. C i t r a t e s y n t h e t a s e was a s s a y e d i n b o t h th e s u p e r n a t a n t and resuspended p e l l e t , and t h e c o n d i t i o n s under which no m i t o c h o n d r i a l breakage o c c u r r e d were: 1. when a n i m a l s were n o t p r e v i o u s l y f r o z e n , and 2. when the b u f f e r c o n t a i n e d 0.01 M T r i s , pH 7.5 and 5 mM Mg . CK a c t i v i t y i n t h e s u p e r -n a t a n t f r a c t i o n was a s s a y e d i n t h e absence o f m i t o c h o n d r i a l breakage. Any change i n s p e c i f i c a c t i v i t y d u r i n g development i s e v i d e n t l y due t o c y t o p l a s m i c CK. The CK a c t i v i t y i n t h e crude m i t o c h o n d r i a l p r e p a r a t i o n r e p r e s e n t e d from z e r o t o 10% o f the t o t a l CK a c t i v i t y ; v e r y l i t t l e o r no CK a c t i v i t y was p r e s e n t i n t h e v e r y e a r l y embryos b u t t h e a c t i v i t y i n c r e a s e d g r a d u a l l y . t o about 10% o f the t o t a l a c t i v i t y a t . s t a g e 42 and 48. P o s s i b l y t h e g r a d u a l i n c r e a s e i n " m i t o c h o n d r i a l " CK a c t i v i t y was an a r t e f a c t o f the homogeniza-t i o n p r o c e d u r e r a t h e r t h a n an a c t u a l i n c r e a s e i n s p e c i f i c 22 a c t i v i t y o f t h e enzyme i n the p e l l e t e d f r a c t i o n . A c y t o p l a s -mic enzyme, LDH, was assayed i n e x t r a c t s , i n w h i c h no mitochond-r i a l breakage o c c u r r e d , and an i n c r e a s e from 0 t o 10% o f the LDH a c t i v i t y was found. I n t h e crude m i t o c h o n d r i a l p r e p a r a t i o n as development proceeded. Thus, i t has not been e s t a b l i s h e d c l e a r l y t h a t t h e i n c r e a s e i n CK a c t i v i t y i n the p e l l e t e d p o r t i o n , o f t h e e x t r a c t i s m i t o c h o n d r i a l CK r a t h e r t h a n an a r t e f a c t o f the h o m o g e n i z a t i o n p r o c e d u r e . A d u l t Isozyme P a t t e r n s : The e l e c t r o p h o r e t i c p a t t e r n s o f c r e a t i n e k i n a s e from v a r i o u s a d u l t organs o f Xenopus a r e shown i n F i g u r e 3. I n a d u l t s k e l e t a l muscle, isozyme I I I p r e d o m i n a t e d , and sometimes t r a c e s o f isozyme I I were a p p a r e n t . Isozyme I was never o b s e r v e d i n any p r e p a r a t i o n s from s k e l e t a l muscle. An i d e n t i c a l isozyme p a t t e r n was o b s e r v e d i n c a r d i a c muscle. I n t h e b r a i n , o n l y isozyme I was o b s e r v e d . The r e s o l u t i o n o f isozyme I i n t o 3 bands was o b s e r v e d i n o n l y one i n d i v i d u a l ; u s u a l l y o n l y 1 band was p r e s e n t i n b r a i n t i s s u e . The stomach showed th e same i s o -zyme p a t t e r n as t h e b r a i n . When g e l s were i n c u b a t e d i n t h e absence o f CP/ no e l e c t r o p h o r e t i c bands were p r e s e n t . Ontogeny o f C r e a t i n e K i n a s e Isozymes: The e l e c t r o p h o r e t i c p a t t e r n s o f u n f e r t i l i z e d eggs and e a r l y embryos are p r e s e n t e d i n F i g u r e 4. Isozyme I was p r e s e n t d u r i n g e a r l y embryogenesis up t o t h e free-swimming l a r v a l s t a g e (42); then isozyme I I appeared. The p a t t e r n remained the same u n t i l a f t e r metamorphosis (stage 66) when isozyme I I I appeared i n 23 F i g u r e 3. Agarose g e l e l e c t r o p h o r e t o g r a m o f c r e a t i n e k i n a s e isozymes o f a d u l t Xenopus l a e v i s o r g a n s . Isozyme I , c h a r a c t e r i s t i c o f b r a i n , i s not n o r m a l l y s p l i t i n t o 3 f r a c t i o n s , a, b, and c. I , I I , I I I r e p r e -s e n t CK isozymes. S k e l e t a l M H e a r t S t o m a c h B ra in 25 F i g u r e 4. C r e a t i n e k i n a s e isozymes o f s e q u e n t i a l d e v e l o p -m ental s t a g e s . Embryos were s t a g e d a c c o r d i n g t o Nieuwkoop and Faber; 0 = O r i g i n . 2 6 III M I S k e l e t a l M u s c l e S t a g e 4 8 S t a g e 4 2 S t a g e 3 3 - 3 4 S t a g e 2 6 - 2 7 S t a g e 10-11 Bra in t o 27 muscle. M i c r o h e t e r o g e n e i t y o f isozymes I and I I was o b s e r v e d i n premetamorphic t a d p o l e s o f s t a g e s 46, 47, 48, and 4 9 which were o b t a i n e d from a s i n g l e m a t i n g ( F i g u r e 5 ) . One i n d i v i d u a l was used f o r e l e c t r o p h o r e s i s a t each s t a g e o f development. The h e t e r o g e n e i t y o b s e r v e d i n t h e s e t a d p o l e s was not n o r m a l l y o b s e r v e d . U s u a l l y o n l y one band appeared i n t h e p o s i t i o n s c o r r e s p o n d i n g t o isozymes I and I I I a t t h e s e d e v e l o p m e n t a l s t a g e s . F i g u r e 6 shows l a t e r d e v e l o p m e n t a l s t a g e s , p a r t i c u l a r l y t a d p o l e s a p p r o a c h i n g metamorphic c l i m a x . The a n i m a l s were from a d i f f e r e n t s e t d f p a r e n t s t h a n t h o s e i n F i g u r e 5. The t r a n s i -t i o n from t h e embryonic t o the a d u l t p a t t e r n was s t i l l n o t a p p a r e n t . E l e c t r o p h o r e t i c p a t t e r n s o b t a i n e d from r e g i o n s o f s t a g e 58 t a d p o l e s a r e d e p i c t e d i n F i g u r e 7. Isozyme I , c h a r a c -t e r i s t i c o f a d u l t b r a i n , was p r e s e n t i n t h e t a d p o l e b r a i n . The back muscle r e g i o n c o n t a i n e d b o t h isozymes I and I I ; whereas the l e g and t a i l muscles c o n t a i n e d o n l y isozyme I I . The s k e l e -t a l muscle from a f r o g w h i c h had j u s t completed metamorphosis showed b o t h isozymes I I and I I I . I n d u c t i o n o f Metamorphosis: Stage 58 t a d p o l e s j u s t a p p r o a c h i n g metamorphic c l i m a x - 8 (emergence o f f o r e l i m b s ) , were t r e a t e d w i t h 1.5 X 10 M T^ f o r 3 days. The r e s u l t s o f t h i s t r e a t m e n t are shown i n F i g u r e 8. I t was apparent t h a t o n l y isozymes I and I I were p r e s e n t i n c o n t r o l and T^ t r e a t e d t a d p o l e s . Only isozyme I I appeared i n the t a i l . The body c o n t a i n e d b o t h isozymes I and I I . Stage 54 t a d p o l e s were t r e a t e d w i t h 3 d i f f e r e n t c o n c e n t r a -28 C r e a t i n e k i n a s e isozymes of t a d p o l e s o f Xenopus  l a e v i s . These animals were a l l progeny o f one s e t o f parents and onl y one animal was used f o r each stage. Isozymes I and I I were r e s o l v e d i n t o 3 f r a c t i o n s , a, b, and c. In t h i s f i g u r e and i n s e v e r a l f o l l o w i n g f i g u r e s , n o n - s p e c i f i c s t a i n i n g i s apparent a t the o r i g i n . T h i s a r t e f a c t i s observed when g e l s are i n c u b a t e d i n the absence o f s u b s t r a t e (CP). 1. A d u l t b r a i n 2. Stage 4 6 3. Stage 47 4. Stage 48 5. Stage 4 9 6. A d u l t s k e l e t a l muscle. 29 + 30 F i g u r e 6. C r e a t i n e k i n a s e isozyme p a t t e r n s o f premetamorphic t a d p o l e s . No t r a n s i t i o n t o t h e a d u l t p a t t e r n c h a r a c t e r i s t i c o f s k e l e t a l muscle was a p p a r e n t . Note t r a c e o f isozyme I I p r e s e n t i n s t a g e 66 s k e l e t a l muscle. 1. A d u l t b r a i n 2. Stage 52 3. Stage 54 4. Stage 57 5. Stage 58 6. Stage 66 s k e l e t a l muscle. 31 32 F i g u r e 7. C r e a t i n e k i n a s e isozymes o f r e g i o n s o f s t a g e 58 t a d p o l e s . 1. Stage 66 b r a i n (post metamorphic f r o g s ) 2. Stage 58 b r a i n 3. Stage 58 "the r e s t " 4. Stage 58 t a i l muscles 5. Stage 58 back muscles 6. Stage 5 8 h i n d l i m b s 7. Stage 66 s k e l e t a l muscle. 33 34 t i o n s o f f o r v a r i o u s t i m e i n t e r v a l s , and the r e s u l t s o f t r e a t m e n t a r e shown i n F i g u r e s 8, 9, and 10. No change i n i s o -zyme p a t t e r n was o b s e r v e d a f t e r 3 days o f t r e a t m e n t w i t h - 8 1.5 X 10 M T3 ( F i g u r e 8); the r e s t o f the t a d p o l e s t r e a t e d t h i s way d i e d w i t h i n 6 days. A n o t h e r b a t c h o f t a d p o l e s was - 9 t r e a t e d w i t h 7.5 X 10 M ; however, t h e y a l l e x p i r e d w i t h i n - 9 5 days. The t h i r d b a t c h was t r e a t e d w i t h 1.5 X 10 M T^, and the CK isozymes o f c o n t r o l and T^ t a d p o l e s a r e shown i n F i g u r e s 9 and 10. A f t e r one week o f t r e a t m e n t , isozymes I and I I were o b s e r v e d i n the body, and o n l y isozyme I I was p r e s e n t i n the t a i l s . The same r e s u l t s a r e o b t a i n e d a f t e r t r e a t i n g a n i m a l s f o r - 9 2 weeks i n 1.5 X 10 M T 3 ( F i g u r e 1 0 ) . The l e g s o f t h e s e t a d -p o l e s were l a r g e enough t o be removed and e l e c t r o p h o r e s e d s e p a r -a t e l y ; however, no t r a n s i t i o n from isozyme I I t o I I I was a p p a r e n t . S i m i l a r l y , a f t e r 21 days o f t r e a t m e n t , no change i n isozyme p a t t e r n was o b s e r v e d ( r e s u l t s n ot shown). W h i l e T^ i n d u c e d some metamorphic changes, i . e . , t a i l and body l e n g t h d e c r e a s e d , l e g l e n g t h i n c r e a s e d , and w e i g h t d e c r e a s -ed r e l a t i v e t o the c o n t r o l s , no CK t r a n s i t i o n was i n d u c e d . 35 F i g u r e 8. C r e a t i n e k i n a s e isozymes o f c o n t r o l and t r e a t e d t a d p o l e s . One s e t o f a n i m a l s was t r e a t e d a t s t a g e 58; t h e o t h e r t r e a t e d a t st a g e 54. Both s e t s o f t r e a t e d t a d p o l e s were t r e a t e d w i t h hormone (1.5 X 1 0 _ 8 M ) f ° r 3 d a y S -1. A d u l t b r a i n 2. Stage 54 b o d y — c o n t r o l 3. Stage 54 b o d y — t r e a t e d w i t h 4. Stage 54 t a i l — t r e a t e d w i t h 5. Stage 58 b o d y — c o n t r o l 6. Stage 58 b o d y — t r e a t e d w i t h 7. Stage 58 t a i l — t r e a t e d w i t h T 3. No isozyme t r a n s i t i o n was a p p a r e n t . 37 F i g u r e 9. C r e a t i n e k i n a s e isozymes o f c o n t r o l and T^ t r e a t e d t a d p o l e s . T a d p o l e s were t r e a t e d a t s t a g e 54 w i t h -9 1.5 X 10 M T3« A f t e r one week on t r e a t m e n t no -isozyme t r a n s i t i o n was d e t e c t a b l e . 1. Stage 66 b r a i n (post metamorphic f r o g ) 2. Stage 54 b o d y — c o n t r o l 3. Stage 54 t . a i l - - c o n t r o l 4. b l a n k 5. Stage 54 b o d y — T ^ t r e a t e d 6. Stage 54 t a i l — T ^ t r e a t e d 7. b l a n k 8. Stage 66 s k e l e t a l muscle. 38 39 F i g u r e 10. C r e a t i n e k i n a s e i s o z y m e s o f c o n t r o l and t r e a t e d t a d p o l e s . T a d p o l e s were t r e a t e d i d e n t i c a l l y t o t h o s e i n F i g u r e 9 , e x c e p t t h e s e t a d p o l e s were t r e a t e d f o r 2 weeks. / 1. B r a i n 2. S t a g e 54 t a i l — c o n t r o l 3. S t a g e 54 b a c k and l e g s — c o n t r o l 4. S t a g e 54 t a i l — T 3 t r e a t e d 5. S t a g e 54 l e g s — T ^ t r e a t e d 6. S t a g e 54 b a c k — T 3 t r e a t e d 7. S t a g e 66 s k e l e t a l m u s c l e . 40 + 41 DISCUSSION I n t h i s s t u d y I examined changes i n c r e a t i n e k i n a s e enzyme a c t i v i t y and t h e t e m p o r a l and s p a t i a l d i s t r i b u t i o n o f isozyme p a t t e r n s i n a d u l t and d e v e l o p i n g Xenopus l a e v i s . Changes i n t h e s p e c i f i c a c t i v i t y o f an enzyme d u r i n g development, changes t h a t can o f t e n be c o r r e l a t e d w i t h s p e c i f i c m o r p h o l o g i c a l e v e n t s and the a c q u i s i t i o n o f new m e t a b o l i c f u n c t i o n s , may r e f l e c t changes i n gene a c t i v i t y . Thus, s p e c i f i c a c t i v i t y o f an enzyme i n d i c a t e s t h e s t a t e o f d i f f e r e n t i a t i o n o f s p e c i f i c c e l l t y p e s . However, changes i n s p e c i f i c a c t i v i t y o f an enzyme may a l s o r e f l e c t o t h e r p a r a m e t e r s , i n c l u d i n g v a r i a t i o n o f background p r o t e i n c o n c e n t r a -t i o n , a change i n i n t r a c e l l u l a r l o c a l i z a t i o n , o r a change i n t h e p r o p e r t i e s o f a p r e - e x i s t i n g enzyme. A more s e n s i t i v e i n d i c a -t i o n o f gene f u n c t i o n i s p r o v i d e d when isozyme changes are m o n i t o r e d as w e l l (Masters and Holmes, 1972). As the r e s u l t s have shown, t h e s p e c i f i c a c t i v i t y o f CK remains r e l a t i v e l y s t a b l e t h r o u g h o u t development u n t i l s t a g e 26/27 when th e s p e c i f i c a c t i v i t y d e c r e a s e s . CK a c t i v i t y b e g i n s t o i n c r e a s e a g a i n a t s t a g e 42 and r i s e s s h a r p l y between s t a g e 4 2 and 48. The change i n s p e c i f i c a c t i v i t y o f CK d u r i n g t h e e a r l y development o f Xenopus may be a r e f l e c t i o n o f a change i n t h e amount o f p r o t e i n e x t r a c t e d a t the v a r i o u s d e v e l o p m e n t a l s t a g e s r a t h e r t h a n a change i n / gene f u n c t i o n . Enzyme a c t i v i t y p e r u n i t o f e x t r a c t a b l e p r o t e i n may n o t be a s u i t a b l e b a s i s o f r e f e r e n c e f o r e x p r e s s i n g changes i n enzyme a c t i v i t y d u r i n g d i f f e r e n t i a -t i o n . One would n o t have p r e d i c t e d a drop i n t h e . s p e c i f i c a c t i v i t y o f CK a t s t a g e 26/27 when the embryo i s f i r s t c a p a b l e o f spontaneous movements s i n c e CK has been shown t o be impor-t a n t f o r ATP g e n e r a t i o n d u r i n g muscle c o n t r a c t i o n (Watts, 1973). I t i s noteworthy t h a t f o r one e x p e r i m e n t , the p e r c e n t a g e o f t o t a l p r o t e i n e x t r a c t e d a t s u c c e s s i v e d e v e l o p m e n t a l s t a g e s was c a l c u l a t e d . As t h e s p e c i f i c a c t i v i t y d e c r e a s e d , t h e p e r c e n t a g e o f t o t a l p r o t e i n w h i c h was e x t r a c t e d by t h e homogenization. p r o -cedure i n c r e a s e d . L i k e w i s e , as t h e s p e c i f i c a c t i v i t y i n c r e a s e d , t h e p e r c e n t a g e o f t o t a l e x t r a c t a b l e p r o t e i n d e c r e a s e d . D i f f e r -e n t i a l e x t r a c t i o n o f p r o t e i n a t v a r i o u s s t a g e s may a c c o u n t f o r some o f t h e changes i n s p e c i f i c a c t i v i t y , even though i d e n t i c a l e x t r a c t i o n p r o c e d u r e s were used a t each s t a g e s t u d i e d . D i f f e r e n t i a l e x t r a c t i o n o f p r o t e i n a t d i f f e r e n t s t a g e s i s not unexpected s i n c e ' d u r i n g embryonic development d i f f e r e n t i a t -i n g c e l l s s y n t h e s i z e s p e c i f i c p r o t e i n s from amino a c i d s s u p p l i e d by t h e breakdown o f y o l k p l a t e l e t s (Selman and Pawsey, 1965). W h i l e t h e t o t a l p r o t e i n o f Xenopus does not change u n t i l s t a g e 45 ( f e e d i n g ) , p r o t e i n i s r e d i s t r i b u t e d from t h e i n s o l u b l e y o l k p l a t e l e t s t o t h e s o l u b l e c e l l u l a r components. The embryo d o u b l e s i n l e n g t h between s t a g e 11 and s t a g e 26, and d o u b l e s a g a i n by s t a g e 33/34 (Nieuwkoop and F a b e r , 1967). The t a i l b u d i s d i s -t i n c t by s t a g e 29, and presumably a number o f s o l u b l e p r o t e i n s are b e i n g s y n t h e s i z e d d u r i n g t h e s e p e r i o d s . I n f a c t , Selman and Pawsey (1965) have shown t h a t r a p i d u t i l i z a t i o n o f y o l k p l a t e -43 l e t s i n Xenopus b e g i n s i n some t i s s u e s between s t a g e s 25 and 29, and a r a p i d r a t e o f y o l k p l a t e l e t u t i l i z a t i o n c o n t i n u e s u n t i l s t a g e 45, a f t e r w h i c h the r a t e o f u t i l i z a t i o n d e c r e a s e s . Whole embryo homogenates were used' f o r t h e a s s a y s i n t h e s e e-xperiments. C o n s e q u e n t l y changes i n s p e c i f i c a c t i v i t y a re d i f f i c u l t t o i n t e r p r e t i n a d e v e l o p m e n t a l l y s i g n i f i c a n t way. R e g i o n a l v a r i a t i o n o f t h e enzyme may g o . u n d e t e c t e d . A l s o , a number o f i n d i v i d u a l s (15-100) , depending on t h e s t a g e o f d e v e l -opment, were r e q u i r e d f o r t h e a s s a y s . I n each e x p e r i m e n t , groups o f embryos from t h e same m a t i n g were u t i l i z e d , and we assume i d e n t i c a l g e n e t i c c o m p o s i t i o n . Changes i n enzyme a c t i v i t y may be i n d i c a t i v e o f a l t e r e d p r o p e r t i e s o f t h e enzyme r a t h e r t h a n changes i n t h e amount p r e s e n t . T h e r e f o r e , o p t i m a l c o n d i t i o n s a t each s t a g e o f d i f f e r -e n t i a t i o n s t u d i e d must be e s t a b l i s h e d as pH, t e m p e r a t u r e , co-f a c t o r and s u b s t r a t e optima may change as development p r o c e e d s . Indeed, L y z l o v a and Guncheva (1973) have found t h a t d u r i n g t h e metamorphosis o f Rana t e m p o r a r i a , t h e t e m p e r a t u r e s e n s i t i v i t y o f the enzyme c r e a t i n e k i n a s e changes. D u r i n g e a r l y d e v e l o p m e n t a l s t a g e s t h e a c t i v i t y wa|; h i g h e r a t 37°C t h a n a t 20°C, but d u r i n g J metamorphosis, t h e t e m p e r a t u r e s e n s i t i v i t y changes, and the a c t i v i t y i s h i g h e r a t 20°C th a n a t 37°C. There was no c h a n g e - i n v / t h e isozyme c o m p o s i t i o n o f the r e g i o n s s t u d i e d d u r i n g the p e r i o d J o f t h e r e v e r s a l o f t e m p e r a t u r e s e n s i t i v i t y . Changes In enzyme a c t i v i t y , w h i c h o c c u r c o n c o m i t a n t l y w i t h changes i n isozyme p a t t e r n , p r e s e n t a d d i t i o n a l d i f f i c u l t i e s o f 44 i n t e r p r e t a t i o n as the assay employed may n o t p r o v i d e o p t i m a l c o n d i t i o n s f o r each isozyme. I n f a c t , most o f t h e isozymes ex-h i b i t d i f f e r e n t k i n e t i c p r o p e r t i e s . Once o p t i m a l a s s a y c o n d i -t i o n s have been e s t a b l i s h e d , o n l y the enzyme i s r a t e - l i m i t i n g , and t h e p o t e n t i a l i t i e s o f . t h e enzyme can be a s s a y e d . Measure-ments o f the t o t a l a c t i v i t y in. v i t r o , under c o n d i t i o n s t h a t t h e enzyme may never e n c o u n t e r , o n l y i n d i c a t e . , the p o t e n t i a l f u n c -t i o n o f the enzyme. The i n v i v o f u n c t i o n o f CK would p r o b a b l y depend on the s u b s t r a t e c o n c e n t r a t i o n r a t h e r t h a n the enzyme c o n c e n t r a t i o n , s i n c e CK i s n o t a r e g u l a t o r y enzyme. Some o f the problems mentioned above may p a r t i a l l y a c c o u n t f o r the v a r i a b i l i t y o b s e r v e d . S i n c e the t e c h n i q u e i s not p r e c i s e , an a t t empt was made t o e s t a b l i s h o n l y d e v e l o p m e n t a l t r e n d s . We can say, t h e r e f o r e , t h a t CK a c t i v i t y , e x p r e s s e d as a f u n c t i o n o f e x t r a c t a b l e p r o t e i n , remains s t a b l e d u r i n g e a r l y development, drops s l i g h t l y when the a n i m a l b e g i n s t o move about (stage 26/27) and i n c r e a s e s a g a i n a t the premetamorphic s t a g e (stage 4 8 ) . P e r h a p s , because the change i n enzyme a c t i v i t y i s so d r a m a t i c a t s t a g e 48, t h i s b i o c h e m i c a l e v e n t can be c o r r e l a t -ed w i t h m o r p h o l o g i c a l changes s i n c e a t t h a t s t a g e the somite m y o b l a s t s o f Xenopus f u s e t o form m u l t i n u c l e a t e - c o n t r a c t i l e myotubes (Muntz, 19 75). The d i s t r i b u t i o n of. CK isozymes i n Xenopus a d u l t t i s s u e s and the ontogeny o f t h e s e isozyme p a t t e r n s has been i n v e s t i g a t -ed. Three major e l e c t r o p h o r e t i c v a r i a n t s a r e a p p a r e n t i n a d u l t and d e v e l o p i n g Xenopus, and some m i c r o h e t e r o g e n e i t y i s o b s e r v e d 45 f o r the major bands. The ontogeny o f t h e isozymes i s anomalous compared t o the ontogeny o f CK i n r a t s and c h i c k e n s . Isozyme I i s p r e s e n t t h r o u g h o u t development, isozyme I I appears a t s t a g e 42, and isozyme I I I i s n o t d e t e c t a b l e u n t i l a f t e r metamorphosis. Isozyme I I i s p r e s e n t i n muscle from t a d p o l e s , a b s e n t i n a d u l t m uscle, and may r e p r e s e n t a t r a n s i e n t , embryonic, m u s c l e - s p e c i f i c form o f CK. Now t h a t the t e m p o r a l and s p a t i a l p a t t e r n o f CK appearance has been e s t a b l i s h e d , i t i s n e c e s s a r y t o d e t e r m i n e the g e n e t i c b a s i s o f isozyme f o r m a t i o n , the p h y s i o l o g y o f the changes, and the c o n t r o l o f isozyme appearance d u r i n g d e v e l o p -ment. U s u a l l y two, and sometimes t h r e e , major e l e c t r o p h o r e t i c bands o f CK o c c u r i n the a d u l t t i s s u e s o f Xenopus. Only isozyme I I I , t h e s l o w e s t m i g r a t i n g enzyme, was n o r m a l l y o b s e r v e d i n s k e l e t a l and c a r d i a c muscle, b u t t r a c e s o f isozyme I I were some-times o b s e r v e d . Isozyme I I was n o t the predominant isozyme i n any a d u l t t i s s u e examined. I t i s p o s s i b l e t h a t the t i s s u e s examined i n t h i s i n s t a n c e were t a k e n from young f r o g s , as t h e r e i s no way t o t e l l the age o f t h e f r o g s o b t a i n e d from South A f r i c a . - The b r a i n and stomach c o n t a i n e d . o n l y isozyme I , which o n l y once was o b s e r v e d t o s p l i t i n t o 3 bands i n a d u l t t i s s u e . A l l 3 isozymes were never o b s e r v e d t o e x i s t t o g e t h e r i n a s i n g l e t i s s u e t y pe i n Xenopus. The r e s u l t s o b t a i n e d f o r Xenopus a d u l t organs a r e s i m i l a r t o those o b t a i n e d by Eppenberger e t a_l. (1964) f o r the a d u l t c h i c k e n and r a t o r g a n s . 4 6 The isozyme p a t t e r n o b s e r v e d i n Xenopus development^ however i s d i f f e r e n t from the r e s u l t s o b t a i n e d by Eppenberger e t al . . (1964) f o r c h i c k e n s . D u r i n g t h e development o f c h i c k e n s k e l e -t a l muscle t h e r e was a g r a d u a l p r o g r e s s i o n from the BB t o t h e MB t o t h e MM isozyme, w i t h a l l 3 isozymes a p p e a r i n g t o g e t h e r i n t h i s t i s s u e t y p e . I n Xenopus development t h e r e appeared t o be no t r a n s i t i o n from isozyme I t o isozyme I I I w i t h isozyme I I b e i n g t h e h y b r i d form o f the enzyme. There was a t r a n s i t i o n from isozyme I t o isozyme I I , b u t i t has not b e e n . e s t a b l i s h e d t h a t t h i s t r a n s i t i o n o n l y o c c u r r e d i n muscle t i s s u e . Isozyme I I , and t r a c e s o f isozyme I , have been l o c a l i z e d i n the s k e l e -t a l muscle o f s t a g e 54 t a d p o l e s . Whole l e g s , t a i l o r back muscle r e g i o n s were examined, and i t i s not c e r t a i n t h a t when t r a c e s o f isozyme I were p r e s e n t they were l o c a l i z e d i n the muscle t i s s u e . Isozyme I may n e v e r be p r e s e n t i n muscle t i s s u e . Then, d u r i n g metamorphosis t h e r e was a t r a n s i t i o n from isozyme I I t o isozyme I I I , s i n c e isozyme I I was p r e s e n t i n h i g h concen-t r a t i o n s i n muscle t i s s u e a t s t a g e 58 and b o t h isozyme I I and I I I were p r e s e n t i n newly metamorphosed f r o g s . Only isozyme I I I was p r e s e n t i n a d u l t s k e l e t a l muscle. These r e s u l t s s u ggest t h a t isozyme I I i n Xenopus i s a t r a n s i e n t , embryonic muscle-s p e c i f i c form o f CK, r a t h e r than a h y b r i d o f isozymes I and I I I as has been o b s e r v e d i n c h i c k e n s and r a t s . No i n f o r m a t i o n i s a v a i l a b l e c o n c e r n i n g the s u b u n i t composi-t i o n o f Xenopus CK, t h e r e f o r e yany a n a l y s i s o f the m o l e c u l a r b a s i s f o r isozyme f o r m a t i o n i s p u r e l y s p e c u l a t i v e . The r e s u l t s suggest that 3 gene l o c i are responsible for the CK isozymes i n Xenopus, and that only one gene locus i s ac t ive i n a s ingle c e l l type. I f CK of Xenopus i s a dimer as i t ex i s t s i n other ver te-bra tes , and i f both genes are ac t ive at the same time, one would expect random as soc ia t ion of subunits as i s observed in. other isozyme systems (Markert, 196 3) with the formation of 3 e l ec t rophore t i c variants.— Since no hybr id i s apparent, the gene fo r I may be turned o f f as the gene for II i s turned on. L i k e -wise, the gene for II i s turned o f f as the gene for III i s turned on. The isozymes would then cons i s t of polypeptides coded by mul t ip le genes to form homodimeric enzymes. I t would be poss ib le to te s t t h i s hypothesis by preparing hybr id forms o f the enzyme i n v i t r o , as has been done for a number of i s o -zyme systems (Markert, 196 3; Eppenberger, 19 75). A l t e r n a t e l y , . the observed e l e c t r o p h o r e t i c var iants could be due to secondary modif icat ions o f the enzyme, such that the e l ec t rophore t i c m o b i l i t y i s s l i g h t l y a l t e red at d i f f e r e n t developmental stages. The microheterogeneity of CK isozymes as seen i n Figure 5 provides a further suggestion that 3 polypeptide subunits under the c o n t r o l of 3 n o n a l l e l i c gene l o c i are responsible for the CK isozyme pattern i n Xenopus. In order to account for the microheterogeneity of isozymes I and II one might make the fo l lowing assumptions: 1. CK i s a dimer', 2. The polypeptide uni t s are under the cont ro l of 2 gene l o c i (one locus for isozyme I and one for 48 isozyme I I ) , 3. Only a l l e l i c p o l y p e p t i d e s h y b r i d i z e t o form an a c t i v e enzyme, 4. A h e t e r o z y g o u s p o p u l a t i o n e x i s t s , 5. Both a l l e l e s o f 2 d i f f e r e n t genes a r e a c t i v e i n a s i n g l e c e l l . I f a l l the above c o n d i t i o n s were s a t i s f i e d , one would o b t a i n the p a t t e r n o b s e r v e d . A l t e r n a t e l y , the o b s e r v e d p a t t e r n c o u l d be e x p l a i n e d by t h e o c c u r r e n c e o f a secondary m o d i f i c a t i o n o f one o f t h e p e p t i d e s i b e f o r e a s s o c i a t i o n i n t o an a c t i v e enzyme m o l e c u l e . Tadpoles e x h i b i t i n g h e t e r o g e n e i t y f o r isozyme I a l s o e x h i b i t e d h e t e r o g e n e i t y f o r isozyme I I s u g g e s t i n g t h a t t h e h e t e r o g e n e i t y i s due t o a p o s t - t r a n s l a t i o n a l m o d i f i c a t i o n . ' I f the h e t e r o g e n e i t y was due t o the e x p r e s s i o n o f h e t e r o z y g o u s a l l e l e s , o n l y one isozyme s h o u l d e x h i b i t h e t e r o g e n e i t y i n a s i n g l e o r g a n i s m . I t s h o u l d be emphasized t h a t i n t h e s e e x p e r i -ments m i c r o h e t e r o g e n e i t y was n o t commonly o b s e r v e d , b u t was o n l y seen i n the a n i m a l s from one m a t i n g and i n the b r a i n and stomach o f one a d u l t . I f a p o p u l a t i o n h e t e r o z y g o u s f o r CK e x i s t s i n Xenopus, one would be a b l e t o d e t e r m i n e t h e c o n t r i b u t i o n s o f the p a r e n t a l a l l e l e s d u r i n g development. H e t e r o z y g o t e s o c c u r r i n g i n a s i n g l e p o p u l a t i o n c o u l d be d e t e c t e d by the presence o f a d d i t i o n a l i s o z y m e s , the number o f w h i c h would depend on the s u b u n i t com-p o s i t i o n o f the enzyme. The o c c u r r e n c e o f m i c r o h e t e r o g e n e i t y o f isozymes i s not uncommon i n Xenopus; Kunz (197 3) and Claycomb and V i l l e e (19 71) have demonstrated t h a t n i n e e l e c t r o p h o r e t i c 49 v a r i a n t s o f LDH e x i s t i n Xenopus. W a l l and B l a c k l e r (1975) o b s e r v e d 16-18 bands o f LDH i n Xenopus, compared t o the u s u a l f i v e o b s e r v e d i n b i r d s and mammals. F u r t h e r work i n t h i s a r e a would i n v o l v e a more p r e c i s e t e m p o r a l and s p a t i a l l o c a l i z a t i o n o f isozyme I I and a d e t e r m i n a -t i o n o f the m o l e c u l a r b a s i s f o r isozyme f o r m a t i o n . I f isozyme I I i s a t r a n s i e n t form o f CK, the p h y s i o l o g i c a l s i g n i f i c a n c e o f the t r a n s i t i o n might p r o v i d e i n f o r m a t i o n r e g a r d i n g s p e c i f i c m e t a b o l i c r e q u i r e m e n t s o f the d e v e l o p i n g embryo. A p o s s i b l e c o r r e l a t i o n e x i s t s between the r i s e i n s p e c i f i c a c t i v i t y o f CK and the appearance o f isozyme I I . I t would a l s o be v e r y i n t e r e s t i n g t o det e r m i n e what causes t h e i n d u c t i o n o f t h i s isozyme d u r i n g development. The t r a n s i t i o n o f isozyme I I t o isozyme I I I presumably o c c u r s d u r i n g metamorphosis o f Xenopus. An at t e m p t was made t o in d u c e the t r a n s i t i o n o f CK from the embryonic form (isozyme I I ) t o the a d u l t s k e l e t a l muscle form (isozyme I I I ) by t r e a t i n g the t a d p o l e s (stage 54 and 58) w i t h t r i i o d o t h y r o n i n e (T-j) • However, isozyme I I I d i d not appear i n the T^ t r e a t e d t a d p o l e s . The f o r m a t i o n o f embryonic p r o t e i n s w h i c h undergo a t r a n s i t i o n t o a d u l t p r o t e i n s d u r i n g metamorphosis o f amphibians i s n o t unique t o the CK i n Xenopus development. L i v e r g l u t a -mate dehydrogenase (Cohen, 1970; Wigg e r t and Cohen, 1966) and hemoglobin (Moss and Ingram, 19 6 8; Herner and F r e i d e n , 1961; F r e i d e n and J u s t , 19 70) undergo s i m i l a r t r a n s i t i o n s d u r i n g the metamorphosis o f Rana c a t e s b e i a n a . I n a d d i t i o n t o t h e s e t r a n s -50 i t i o n s a number o f o t h e r b i o c h e m i c a l changes o c c u r . d u r i n g metamorphosis, i n c l u d i n g a change i n DNA, RNA, and p r o t e i n s y n t h e s i s ( T a t a , 1968, 1971; Weber, 1967) and an i n c r e a s e i n the a c t i v i t y o f a number o f l i v e r enzymes i n v o l v e d i n u r e a b i o s y n t h e s i s (Cohen, 1970; B a l i n s k y e t a l . , 1970). The p r e c o c i o u s appearance o f t h e s e changes can be i n d u c e d by t r e a t -i n g t h e a n i m a l s w i t h exogenous t h y r o i d hormone. The o n l y s p e c i f i c b i o c h e m i c a l i n d u c t i o n s i n response t o exogenous t h y r o i d hormone i n Xenopus have been the p r e c o c i o u s appearance o f a d u l t k e r a t i n - l i k e p r o t e i n s (Reeves, 1976) and carbamoylphosphate s y n t h e t a s e ( B a l i n s k y e t a l . , 1972). D u r i n g the normal development o f Xenopus, k e r a t i n - l i k e p r o t e i n s f i r s t appear around s t a g e 55, b e f o r e metamorphic c l i m a x , and i n c r e a s e about 5 0 - f o l d by s t a g e 66, by the end o f metamorphosis (Reeves, 1975). A t l e a s t a 1 0 - f o l d i n c r e a s e o f immunoreactive a d u l t k e r a t i n - l i k e p r o t e i n s i s o b s e r v e d i n s t a g e 50/52 t a d p o l e s -7 -9 t r e a t e d w i t h 10 M t o 10 M T 3 f o r 4 days (Reeves, 1976). A l s o d u r i n g normal metamorphosis t h e a c t i v i t y / w e t w e i g h t o f c a r b a -moylphosphate s y n t h e t a s e , the r a t e - l i m i t i n g enzyme i n urea f o r m a t i o n , i n c r e a s e s a t s t a g e 62-63 ( B a l i n s k y e t a l . , 1972). D u r i n g i n d u c e d metamorphosis the s p e c i f i c a c t i v i t y o f carbomoyl-phosphate s y n t h e t a s e doubles a f t e r 5 days o f t r e a t m e n t o f stage 57/58 t a d p o l e s w i t h t h y r o x i n e ( B a l i n s k y e t a l . , 1972). I t i s not c l e a r , however, i f t h e r e i s an i n c r e a s e i n the amount o f enzyme o r a d e c r e a s e i n the w e i g h t o f t h e l i v e r . K i s t l e r and Weber (1975) have shown t h a t d u r i n g i n d u c e d metamorphosis t h e r e 51 i s a t r a n s i e n t i n c r e a s e i n l i v e r wet w e i g h t f o l l o w e d by a con-s i d e r a b l e d e c r e a s e . Though a d i r e c t comparison o f t h e r e s u l t s i s n o t p o s s i b l e , an apparent i n c r e a s e i n s p e c i f i c a c t i v i t y c o u l d r e s u l t from l i v e r d e h y d r a t i o n . The d a t a i n t h i s t h e s i s show t h a t t h e CK isozymes i n t h e muscle form o f s t a g e 54 t a d p o l e s do n o t undergo a t r a n s i t i o n t o the a d u l t muscle form d u r i n g i n d u c e d metamorphosis. The f a i l -u r e o f T^ t o i n d u c e the isozyme change c o u l d be caused by s e v e r -a l f a c t o r s . The a n i m a l s may have been t e s t e d f o t an i n s u f f i c i -e n t t i me p e r i o d o r t h e dose o f hormone may. not have been optimum However, w i t h t h e c o n c e n t r a t i o n s o f T^ used i n t h e s e e x p e r i m e n t s the m o r t a l i t y r a t e was v e r y h i g h , and o n l y 10-15 a n i m a l s were used f o r each e x p e r i m e n t . No t a d p o l e s completed metamorphosis. I f more a n i m a l s were a v a i l a b l e , a few might have s u r v i v e d t h r o u g h t h e c o m p l e t i o n o f metamorphosis and the CK isozyme t r a n s i t i o n d e t e c t e d . T e r m i n a l muscle c e l l d i f f e r e n t i a t i o n o c c u r s r e l a t i v e l y l a t e i n Xenopus development, u n l i k e t e r m i n a l e p i d e r m a l c e l l d i f f e r e n t i a t i o n . The a d u l t form o f muscle CK was not d e t e c t e d as l a t e as s t a g e 58, whereas the a d u l t . k e r a t i n -l i k e p r o t e i n s were p r e s e n t a t s t a g e 55 (Reeves, 1975).. The a b i l i t y t o i n d u c e a change i n one system and n o t i n a n o t h e r may be a f u n c t i o n o f t h e normal time c o u r s e o f d e v e l o p m e n t a l e v e n t s . Moreover, i t i s p o s s i b l e t h a t t h e isozyme change i s i n -dependent o f t h y r o i d hormone, as appears t o be the case f o r t h e change from embryonic t o a d u l t hemoglobins d u r i n g Xenopus meta-52 morphosis (Maclean and T u r n e r , 1976). Muscle c e l l s o f Xenopus t a k e on the appearance o f a d u l t s k e l e t a l muscle . r e l a t i v e l y l a t e d u r i n g development. A c c o r d i n g t o Muntz (1975), the myotome muscles resemble a d u l t s k e l e t a l muscle a t s t a g e s 50 t o 55, and t h e h i n d l i m b muscle does not appear f u l l y d i f f e r e n t i a t e d u n t i l s t a g e s 60 t o 61. These ob-s e r v a t i o n s are c o n s i s t e n t w i t h t h o s e o f Kunz (1973); namely, t h a t t h e h i n d l i m b muscles are f u l l y d i f f e r e n t i a t e d by s t a g e 60. The b i o c h e m i c a l r e s u l t s o b t a i n e d i n t h i s t h e s i s are s u p p o r t e d by the m o r p h o l o g i c a l o b s e r v a t i o n s t h a t muscle c e l l s do not a t t a i n a d u l t c h a r a c t e r i s t i c s u n t i l l a t e i n development, d u r i n g the t r a n s i t i o n o f the t a d p o l e t o t h e a d u l t f r o g . A number o f assumptions have been made i n an attempt t o c o r r e l a t e the d a t a i n t h i s t h e s i s w i t h r e p o r t e d o b s e r v a t i o n s on o t h e r s p e c i f i c p r o t e i n changes o c c u r r i n g d u r i n g amphibian meta-morphosis. A change i n the e l e c t r o p h o r e t i c m o b i l i t y o f CK isozymes o c c u r s a t some time between s t a g e s 5 8 and 6 6 i n t h e muscle t i s s u e o f Xenopus. F u r t h e r s t u d i e s on the t i m e , l o c a t i o n and s i g n i f i c a n c e o f the isozyme change, t h e g e n e t i c , b a s i s o f isozyme I I I f o r m a t i o n and whether o r n o t the change i s r e s p o n s -i v e t o hormone a d m i n i s t r a t i o n may p r o v i d e a d d i t i o n a l i n f o r m a t i o n w i t h r e g a r d s t o the c o n t r o l o f g e n e t i c e x p r e s s i o n d u r i n g the development o f t h e t a d p o l e t o t h e f r o g . The r e l e a s e o f hormone s e r v e s t o t r i g g e r a p r e d e t e r m i n e d s e t o f changes; "already d i f f e r e n t i a t e d c e l l s a c q u i r e c h a r a c t e r -i s t i c s ( T a t a , 1971). The l i v e r , b l o o d , and muscle c e l l s a r e 53 r e c o g n i z a b l e as su c h , and metamorphosis o n l y i n v o l v e s a change i n t h e p a t t e r n o f gene e x p r e s s i o n . W h i l e u n d e r s t a n d i n g hormon-a l c o n t r o l o f gene e x p r e s s i o n i s i m p o r t a n t t o t h e problem o f t e r m i n a l c e l l d i f f e r e n t i a t i o n , i t i s e q u a l l y i n t e r e s t i n g t o de t e r m i n e how t h e g e n e t i c e x p r e s s i o n i s c o n t r o l l e d d u r i n g e a r l i e r s t a g e s o f embryogenesis, from t h e f e r t i l i z e d egg t o the t a d p o l e . I n " t h i s c a s e , i t would be i n t e r e s t i n g t o know what i n d u c e s the appearance o f isozyme I I o f c r e a t i n e k i n a s e a t st a g e 42. The d e v e l o p m e n t a l p r o g r e s s i o n o f isozyme I t o i s o -zyme I I t o isozyme I I I s u g g e s t s t h a t t h e b i o c h e m i c a l d i f f e r e n t i -a t i o n o c c u r r i n g d u r i n g e a r l y development o f the u n f e r t i l i z e d egg t o the t a d p o l e i n v o l v e s a g e n e t i c e x p r e s s i o n d i f f e r e n t from t h a t o c c u r r i n g i n the b i o c h e m i c a l d i f f e r e n t i a t i o n from the t a d p o l e t o t h e a d u l t f r o g . F u r t h e r s t u d i e s o f t h e ontogeny o f c r e a t i n e k i n a s e i n Xenopus may p r o v i d e i n f o r m a t i o n o f g e n e t i c and e p i g e n e t i c c o n t r o l o f p r o t e i n s y n t h e s i s d u r i n g development, because CK undergoes t i s s u e - and s t a g e - s p e c i f i c changes d u r i n g embryogene-s i s . The t r a n s i t i o n from the embryonic t o t h e a d u l t p a t t e r n i n muscle i s i n d i c a t i v e o f t e r m i n a l muscle c e l l d i f f e r e n t i a t i o n . I n Xenopus t h e CK t r a n s i t i o n from t h e embryonic t o t h e a d u l t p a t t e r n o c c u r s d u r i n g metamorphosis, and t h i s t r a n s i t i o n may p r o v i d e a u s e f u l system f o r s t u d y i n g the hormonal c o n t r o l o f gene e x p r e s s i o n . 54 SUMMARY 1. S p e c i f i c a c t i v i t y of c r e a t i n e k i n a s e (CK) i n Xenopus  l a e v i s remains co n s t a n t through e a r l y development and i n -c r e a s e s between stages 42 and 48 (swimming t a d p o l e ) . 2. The CK isozyme p a t t e r n s o f a d u l t organs o f Xenopus r e v e a l " the e x i s t e n c e o f 2 major e l e c t r o p h o r e t i c v a r i a n t s . An, a n o -d i c form (isozyme I) i s c h a r a c t e r i s t i c o f a d u l t b r a i n and stomach, and a s i n g l e c a t h o d i c form (isozyme I I I ) i s c h a r a c -t e r i s t i c o f a d u l t s k e l e t a l and c a r d i a c muscle. A t r a c e of an i n t e r m e d i a t e form, isozyme I I , i s observed o c c a s i o n a l l y i n a d u l t organs. 3. The isozyme p a t t e r n remains unchanged from the e a r l i e s t developmental stage s t u d i e d ( u n f e r t i l i z e d eggs) through stage, 33/34 ( h e a r t b e a t ) . In these stages, o n l y the anodic isozyme (isozyme I) i s p r e s e n t . 4. Isozyme I I i s d e t e c t a b l e a t stage 42, and i s l o c a l i z e d i n the s k e l e t a l muscle o f premetamorphic tad p o l e s (stage 54 and 53) and post-metamorphic f r o g s (stage 66). 5. Isozyme I I I , the isozyme c h a r a c t e r i s t i c o f a d u l t muscle, i s f i r s t d e t e c t e d i n the l e g muscle of post-metamorphic f r o g s (stage 66). An attempt to induce the p r e c o c i o u s appearance o f t h i s isozyme d u r i n g metamorphosis u s i n g t r i i o d o t h y r o n i n e has not been s u c c e s s f u l . 55 REFERENCES B a l i n s k y , J . B., G. E. Shambaugh I I I , and P. P. Cohen. 1970. Glutamate Dehydrogenase B i o s y n t h e s i s i n Amphibian L i v e r P r e p a r a t i o n s . J . B i o l . Chem. 245: 128-137. B a l i n s k y , J . B., T. L. C o e t z e r , and F. J . M a t t h e y s e . 1972. The E f f e c t o f T h y r o x i n e and H y p e r t o n i c Environment on t h e Enzymes o f t h e Urea C y c l e i n Xenopus l a e v i s . Comp. Biochem. P h y s i o l . 43B: 83-95. Benbow, R. M., R. Q. W. P e s t e l l , and C. C. F o r d . 1975. Appear-ance o f DNA Polymerase A c t i v i t i e s d u r i n g E a r l y Development o f XenOpus l a e v i s . Dev. B i o l . 43: 159-174. Brown, D., and J . D. C a s t o n . 1962. B i o c h e m i s t r y o f Amphibian Development I . Ribosome and P r o t e i n S y n t h e s i s i n E a r l y Development o f Rana p i p i e n s . Dev. B i o l . 5: 412-434. Champion, M. J . , and G. S. W h i t t . 1976. D i f f e r e n t i a l Gene E x p r e s s i o n i n M u l t i l o c u s Isozyme Systems o f t h e Develop-i n g Green S u n f i s h . J . Exp. Z o o l . 196: 263-282. Claycomb, W. C., and C. A. V i l l e e . 1971. L a c t a t e Dehydrogen-ase Isozymes o f Xenopus l a e v i s : F a c t o r s . A f f e c t i n g T h e i r Appearance d u r i n g E a r l y Development. Dev. B i o l . 24: 413-427. Cohen, P. P. 1970. B i o c h e m i c a l D i f f e r e n t i a t i o n d u r i n g Amphib-i a n Metamorphosis. S c i e n c e 168: 533-536. Dawson, D. M., and H. M. Eppenberger. 1970. C r e a t i n e K i n a s e . Methods Enzymol. 17A: 995-1002. Dawson, D. M., H. M. Eppenberger, and N. 0. K a p l a n . 1965. C r e a t i n e K i n a s e : E v i d e n c e f o r a D i m e r i c S t r u c t u r e . Biochem. B i o p h y s . Res. Comm. 21: 346-353. Dawson, D. M., H. M. Eppenberger, and N. 0. K a p l a n . 1967. The Comp a r a t i v e Enzymology o f C r e a t i n e K i n a s e s I I . P h y s i c a l and C h e m i c a l P r o p e r t i e s . J . B i o l . Chem. 242: 210-217. Deuchar, E. M. 1975. Xenopus: The South A f r i c a n Clawed F r o g . John W i l e y and Sons, London. 354 pp. de V e l l i s , J . 1972. Enzyme R e g u l a t i o n d u r i n g C e l l D i f f e r e n t i a -t i o n . Pages 284-311 i n 0. A. S c h l e i d e and J . de V e l l i s , eds. C e l l D i f f e r e n t i a t i o n . Van N o s t r a n d R e i n h o l d Co., New Yo r k . 56 E p p e n b e r g e r , H. M. 1975. The C o n t r o l o f Isozyme P a t t e r n s i n A n i m a l D e v e l o p m e n t . Pages 217-257 i n R. Weber, e d . The  B i o c h e m i s t r y o f A n i m a l D e v e l o p m e n t . A c a d e m i c P r e s s , New Y o r k . E p p e n b e r g e r , H. M., M. E p p e n b e r g e r , R. R i c h t e r i c h , and H. A e b i . 1964. The O n t o g e n y o f C r e a t i n e K i n a s e I s o e n z y m e s . Dev. B i o l . 10: 1-16. E p p e n b e r g e r , H. M., D.M. Dawson, and N. O. K a p l a n . 1967. The C o m p a r a t i v e E n z y m o l o g y o f C r e a t i n e K i n a s e s I . I s o l a t i o n a nd C h a r a c t e r i z a t i o n f r o m C h i c k e n and R a b b i t T i s s u e s . J . B i o l . Chem. 242: 204-209. E p p e n b e r g e r , H. M., T. W a l l i m a n n , H. J . Kuhn, and D. C. T u r n e r . 1975. L o c a l i z a t i o n o f C r e a t i n e K i n a s e Isozymes i n M u s c l e C e l l s : P h y s i o l o g i c a l S i g n i f i c a n c e . Pages 409-424 i n C. L. M a r k e r t , e d . I s o z y m e s II': P h y s i o l o g y and F u n c t i o n . \ A c a d e m i c P r e s s , New Y o r k . F r e i d e n , E . , and J . J . J u s t . 1970. Hormonal R e s p o n s e s i n A m p h i b i a n M e t a m o r p h o s i s . P a g e s 1-112 i n G. L i t w a c k , e d . B i o c h e m i c a l A c t i o n s o f Hormones, V o l . 1. A c a d e m i c P r e s s , New Y o r k . F r i t z , P. J . , L. W h i t e , and K. M. P r u i t t . 1975. I n t r a c e l l u l a r T u r n o v e r o f L a c t a t e D e h y d r o g e n a s e I s o z y m e s . P a g e s 347-358 i n C. L. M a r k e r t , ed. I s o z y m e s I I I : D e v e l o p m e n t a l  B i o l o g y . A c a d e m i c P r e s s , New Y o r k . G u r d o n , J . B. 1967. A f r i c a n C l a w e d T o a d s . Pages 75-84 i n F. H. W i l t and N. K. W e s s e l s , e d s . Methods i n D e v e l o p m e n t - a l B i o l o g y . Thomas Y. C r o w e l l Co., London. H e r n e r , A. E . , and E. F r e i d e n . 1961. B i o c h e m i c a l Changes d u r -i n g A n u r a n M e t a m o r p h o s i s V I I I . Changes i n t h e N a t u r e of Red C e l l P r o t e i n s . A r c h . B i o c h e m . B i o p h y s . 95: 25 - 3 5 . J a c o b u s , W. E . , and A. L. L e h n i n g e r . 1973. C r e a t i n e K i n a s e o f R a t H e a r t M i t o c h o n d r i a : C o u p l i n g o f C r e a t i n e P h o s p h o r y l a -t i o n t o E l e c t r o n T r a n s p o r t . J . B i o l . Chem. 248: 4803-• 4810. K e l l e r , J . M., and M. N a m e r o f f . 1974. I n d u c t i o n o f C r e a t i n e P h o s p h o k i n a s e i n C u l t u r e s o f C h i c k S k e l e t a l M y o b l a s t s w i t h o u t C o n c o m i t a n t C e l l F u s i o n . D i f f e r e n t i a t i o n 2: 19-23. . K i s t l e r , A., a n d R. Weber. 1975. A Combined B i o c h e m i c a l and M o r p h o m e t r i c S t u d y o f T i s s u e Changes i n Xenopus L a r v a e d u r i n g I n d u c e d M e t a m o r p h o s i s . M o l . C e l l . E n d o c r i n . 2: 261-288. 5 7 Kunz, Y. W. 1973. Changes i n L a c t a t e Dehydrogenase Isozyme P a t t e r n d u r i n g t h e Development o f Xenopus l a e v i s ( Daudin). Revue S u i s s e Z o o l . 80: 431-446. L e b h e r z , H. G. 1975a. Ontogeny and . R e g u l a t i o n o f F r u c t o s e D i -phosphate A l d o l a s e Isoenzymes i n "Red" and "White" S k e l e t a l M u s c l e s o f t h e C h i c k . J . B i o l . Chem. 250: 5976-5981. L e b h e r z , H. G. 1975b. E v i d e n c e f o r t h e Lack o f S u b u n i t Ex-change Between A l d o l a s e T e t r a m e r s i n v i v o . J . B i o l . Chem. 250 (18) : 7388-7391. Lowry, 0. H., N. J . Rosebrough, A. L. F a r r , and R. J . R a n d a l l . 1951. P r o t e i n Measurement w i t h t h e F o l i n P h e n o l Reagent. J . B i o l . Chem. 193: 265-275. L y z l o v a , S. N. and Z. S. Guncheva. 1973. C r e a t i n e K i n a s e o f D e v e l o p i n g T a d p o l e s o f t h e F r o g Rana t e m p o r a r i a . J . E v o l . Biochem. P h y s i o l . 9: 32-36. M a c l e a n , N. and S. T u r n e r . 1976. A d u l t Haemoglobin i n Develop-m e n t a l l y R e t a r d e d T a d p o l e s o f Xenopus l a e v i s . J . Embryol. Exp. Morph. 35: 261-266. M a r k e r t , C. L. and F. M o l l e r . 1959. M u l t i p l e Forms o f Enzymes: T i s s u e O n t o g e n e t i c and S p e c i e s S p e c i f i c . P a t t e r n s . P r o c . Nat. Acad. S c i . 45: 753-763. M a r k e r t , C. L. and H. U r s p r u n g . 1962. The Ontogeny o f Isozyme P a t t e r n s o f L a c t a t e Dehydrogenase i n t h e Mouse. Dev. B i o l . 5: 363-381. . M a r k e r t , C. L. E p i g e n e t i c C o n t r o l o f S p e c i f i c P r o t e i n S y n t h e s i s i n D i f f e r e n t i a t i n g C e l l s . Pages 65-84 i n M. L o c k e , ed. C y t o d i f f e r e n t i a t i o n and M a c r o m o l e c u l a r S y n t h e s i s . Academic P r e s s , New Y o r k . M a s t e r s , C. J. and R. S. Holmes. 1972. Isozymes and Ontogeny. B i o l . Rev. 47: 309-361. M i t c h i s o n , J . M. 1 9 7 1 . The B i o l o g y o f . t h e C e l l C y c l e . Cam-b r i d g e U n i v e r s i t y P r e s s , London. 3 1 3 pp. M o r r i s o n , J . F. and E. James. 1 9 6 5 . The Mechanism o f the R e a c t i o n C a t a l y z e d by A d e n o s i n e T r i p h o s p h a t e - C r e a t i n e Phos-p h o t r a n s f e r a s e . Biochem. J . 9 7 : 3 7 - 5 2 . Moss, B. and V . M. Ingram. 1 9 6 8 . Hemoglobin S y n t h e s i s d u r i n g A m p h i b i a n Metamorphosis I I . S y n t h e s i s o f A d u l t Hemoglobin F o l l o w i n g T h y r o x i n e A d m i n i s t r a t i o n . J . M o l . B i o l . 3 2 : 4 9 3 - 5 0 4 . 58 Muntz, L. 1975. Myogenesis i n t h e Trunk and Leg d u r i n g D e v e l -opment o f t h e Tadpole o f Xenopus l a e v i s ( D a u d i n ) . J. E m b r y o l . Exp. Morph. 33: 757-774. A Nieuwkoop, P. D. and J . F a b e r . 1967. Normal T a b l e s o f Xenopus  l a e v i s ( D a u d i n ) . N o r t h - H o l l a n d P u b l i s h i n g Co., Amsterdam. Reeves, 0. R. 1975. A d u l t Amphibian E p i d e r m a l P r o t e i n s : B i o c h e m i c a l C h a r a c t e r i z a t i o n and Developmental Appearance. J . E m b r y o l . Exp. Morph. 34: 55-73. Reeves, 0. R. 1976. P e r s o n a l Communication. Rugh, R. 1965. E n d o c r i n e F a c t o r s i n Development: E f f e c t o f T h y r o i d Hormone and o f I o d i n e on Amphibian Metamorphosis. Pages 315-319 i n E x p e r i m e n t a l Embryology. Burgess P u b l i s h -i n g Co., M i n n e a p o l i s . S e g e l , I. H. 1975. Enzyme K i n e t i c s . John W i l e y and Sons, New Yo r k . 1297 pp. Selman, G. G. and G. J . Pawsey. 1965. The U t i l i z a t i o n o f Y o l k P l a t e l e t s by T i s s u e s o f Xenopus Embryos S t u d i e d by a S a f r a n i n S t a i n i n g Method. J . Embryol. Exp. Morph. 14: 191-212. S h a i n b e r g , A., G. Y a g i l , and D. Y a f f e . . 1971. A l t e r a t i o n s o f E n z y m a t i c A c t i v i t i e s d u r i n g M u s c l e D i f f e r e n t i a t i o n i n v i t r o . Dev. B i o l . 25: 1-29. S r e r e , P. A. 1969. C i t r a t e S y n t h a s e . Methods Enzymol. 13: 3-16. S t o r e y , K. B. 1974. M e t a b o l i c Consequences o f D i v i n g : The A n o x i c T u r t l e . Ph.D. T h e s i s . • U n i v e r s i t y o f B r i t i s h C o l u m b i a . Vancouver, B. C. Canada. T a t a , J . R. 1968. E a r l y Metamorphic Competence o f Xenopus • L a r v a e . Dev. B i o l . 18: 415-440. T a t a , J . R. 1971. P r o t e i n S y n t h e s i s d u r i n g Amphibian Metamor-p h o s i s . Pages 79-110 i n A. A. Monroy and A. Moscona, eds. C u r r e n t T o p i c s i n Developmental B i o l o g y . Volume 6. Academic P r e s s , New York. T u r n e r , D. C., V. M a i e r , and H. M. Eppenberger. 1974. C r e a t i n e K i n a s e and A l d o l a s e Isoenzyme T r a n s i t i o n s i n C u l t u r e s o f C h i c k S k e l e t a l M u s c l e C e l l s . Dev. B i o l . 37: 63-89. T u r n e r , D.C. 1975. Isozyme T r a n s i t i o n s o f C r e a t i n e K i n a s e and A l d o l a s e d u r i n g M u s c l e D i f f e r e n t i a t i o n i n v i t r o . Pages 145-158 i n C. L. M a r k e r t , ed. Isozymes I I I : D evelopmental  B i o l o g y . Academic P r e s s , New York. 59 Turner, D. C., R. Gmur, H. G. Lebherz, M. S e i g r i s t , T. Walliman, and H. M. Eppenberger. 1976. D i f f e r e n t i a t i o n i n Cultures Derived from Embryonic Chicken Muscle I I . Phosphorylase Histochemistry and Fluorescent Antibody Staining f o r Creatine Kinase and Aldolase. / Dev. B i o l . 48: 284-307. Wall, D. A. and A. W. Bla c k l e r . 1974. Expression of Lactate Dehydrogenase Phenotypes i n I n t r a s p e c i f i c and I n t e r s p e c i f i c Matings of Two Species of Xenopus. Dev. B i o l . 41: 97-109. Watts, D. C. 1973. Creatine Kinase (Adenosine 5'-Triphosphate-Creatine Phosphotransferase). Pages 384-431 i n P. D. Boyer, ed. The Enzymes VIII. Academic Press, New York. Weber, R. 1967. Biochemistry of Amphibian Metamorphosis. Pages 227-301 i n R. Weber, ed. Biochemistry of Animal Development, Volume 2. Academic Press, New York. Whitt, G. S. 1975. isozymes and Developmental Biology. Pages 1-18 i n C. L. Markert, ed. Isozymes I I I : Developmental  Biology. Academic Press, New York. Wiggert, B. 0. and P. P. Cohen. 1966. Comparative Study of Tadpole and Frog Glutamate Dehydrogenases. J . B i o l . Chem. 241: 210-216. 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0094011/manifest

Comment

Related Items