15. 12. 1975

Specialia

c u r v e (Figure 1) were e l e c t r o p h o r e s e d , a n d e n z y m e p a t t e r n s d e l i n e a t e d b y t h e t e t r a z o l i u m procedure. Figure 2 shows some c o m p a r i s o n s of t h e i n v e r t a s e - l i k e a c t i v i t y f o u n d in p r e p a r a t i o n s b y use of t h e t e t r a z o l i u m r e a g e n t . O n l y f a i n t a m o u n t s of a c t i v i t y were o b s e r v e d f r o m m i d log p h a s e p r e p a r a t i o n s . H o w e v e r , invertase-like a c t i v i t y was f o u n d in s t e a d i l y increasing p r o p o r t i o n s as g r o w t h of t h e SL-1 c u l t u r e s p r o g r e s s e d into t h e e a r l y s t a t i o n a r y p h a s e or b e g i n n i n g of t h e s t a t i o n a r y phase. To p e r m i t f u r t h e r c h a r a c t e r i z a t i o n , t h e i n v e r t a s e - l i k e e n z y m e was s e p a r a t e d f r o m o t h e r s u c r o s e - m e t a b o l i z i n g e n z y m e s b y c h r o m a t o g r a p h y of t h e e x t r a c e l l u l a r p r o t e i n p r e p a r a t i o n s on S e p h a d e x G-100. The r e c o v e r e d a c t i v i t y

Fig. 2. Polyacrylamide gel electrophoresis showing invertase activity fl:om extraeellular protein preparations. Samples were from A) late log phase B) early stationary phase mid C) stationary phase.

1401

r e p r e s e n t e d a b o u t 8% of t h e t o t a l s u c r o s e - m e t a b o l i z i n g e n z y m e a c t i v i t y f o u n d p r i o r to t h e c h r o m a t o g r a p h y . The e n z y m e a c t e d on sucrose as s u b s t r a t e to p r o d u c e r e d u c i n g sugar, 49.4% of w h i c h was f o u n d t o be glucose. No p o l y s a c e h a r i d e s y n t h e s i s could be d e m o n s t r a t e d . The e n z y m e h a d a p H o p t i m u m of 6.2, t e m p e r a t u r e o p t i m u m of 38 39 ~ a n d molecular w e i g h t of 48,000. Since trehalose, an e-glucoside, did n o t serve as s u b s t r a t e , t h e e n z y m e was n o t an e-glucosidase. I n t r a c e l l u l a r i n v e r t a s e p r e p a r a t i o n s also g e n e r a t e d e q u i m o l a r f r u c t o s e a n d glucose f r o m sucrose w i t h o u t p o l y s a c c h a r i d e s y n t h e s i s , a n d did n o t g e n e r a t e r e d u c i n g sugar f r o m trehalose. Intracellular invertase preparations and extracellular p r o t e i n p r e p a r a t i o n s were c o m p a r e d e l e c t r o p h o r e t i c a l l y u n d e r several d i f f e r e n t c o n d i t i o n s (5% gel c o n c e n t r a t i o n s a t p H 6.7, 7.3 a n d 8.5; 7% gel c o n c e n t r a t i o n s a t p H 8.5). A t y p i c a l result is s h o w n in F i g u r e 3. In e v e r y i n s t a n c e b o t h intracellular i n v e r t a s e a n d t h e i n v e r t a s e - l i k e e n z y m e in t h e e x t r a c e l l u l a r p r o t e i n p r e p a r a t i o n s p r o d u c e d single a c t i v i t y b a n d s w h i c h h a d t h e s a m e m i g r a t i o n rates. Thus, t h e e n z y m e c h a r a c t e r i s t i c s of t h e i n v e r t a s e - l i k e e n z y m e f r o m t h e c u l t u r e fluids were v e r y similar t o t h o s e for intracellular invertase, as f o u n d in t h i s s t u d y or as p r e v i o u s l y r e p o r t e d a for s t r a i n SL-1. The d a t a t h e r e f o r e i n d i c a t e d t h a t t h e invertase-like e n z y m e is i n t r a c e l l u l a r i n v e r t a s e w h i c h is released i n t o culture fluids p r i m a r i l y d u r i n g lace log a n d s t a t i o n a r y p h a s e s of g r o w t h 1~.

Summary. l n v e r t a s e f r o m e x t r a c e l l u l a r c u l t u r e fluids of S. m~tans strain SL-1 was s h o w n to h a v e t h e s a m e c h a r a c t e r i s t i c s as intracellular i n v e r t a s e f r o m t h e s a m e strain. The d a t a i n d i c a t e t h a t intracellular i n v e r t a s e is released into t h e culture fluids p r i m a r i l y d u r i n g t h e late log a n d s t a t i o n a r y p h a s e s of g r o w t h . R. M. OSBORNE, B. L. L~MB~RTS a n d A. H. R o u s n

Naval Dental Research Institute, Great Lakes (Illinois 60088, USA), and the Department o/ Biology, Illinois Institute o/ Technology, Chicago (Illinois 60616, USA), 20 January I975.

Fig. 3.5 % gel eIectrophoresis of A) extracellular protein preparation and 13) intracellular invertase preparation at pH 7.3. Migration is to the right toward the (+) electrode and enzyme components were delineated by the tetrazolium procedure. The two components with the same migration rate near the right edge of the figure are invertase. Similar results were obtained under other dectrophoretic coBditions.

The authors are grateful to Mr. ]~RNEST PE1)ERSON and Ms. KAREN BUCK, Naval Dental Research Institute, Great Lakes, Illinois for technical assistance. This study constitutes partial fulfillment by RONALO M. OSBORNE of the Master of Science degree requirements of the School of Graduate Studies, Illinois Institute of Technology, Chicago, Illinois. From Research Project No. MRO41.20.02 6048B311, Bureau of Medicine and Surgery, U.S. Navy Department, Washington, D.C. The opinions expressed herein are those of the authors and are not to be construed as reflecting the views of the Navy Department or the Naval Service at large. The use of commercially available products does not imply endorsement of these products or preference to other similar products on the market.

P e n e t r a t i o n of P h o s p h o l i p a s e s A 2 and C into the Squid P h o s p h o l i p a s e A 2 a n d C (PhA~, PhC) are used as e n z y m a t i c probes, to s t u d y t h e s t r u c t u r a l o r g a n i z a t i o n of biological m e m b r a n e s a n d to s e a r c h for specific f u n c t i o n s of p h o s p h o l i p i d s (see 1 for references). I n s t u d i e s on axons, h o w e v e r , c o n n e c t i v e tissue, S e h w a n n cell a n d m y e l i n m a y i n t e r f e r e w i t h t h e access of e x t e r n a l l y applied p h o s p h o lipases to t h e a x o l e m m a w h e r e processes a s s o c i a t e d w i t h

(Loligo pealii)

Giant A x o n

g e n e r a t i o n of t h e a c t i o n p o t e n t i a l occur. L a c k of an effect following t h e a p p l i c a t i o n of p h o s p h o l i p a s e s m a y reflect a n o n - i n v o l v e m e n t of p h o s p h o l i p i d s or m a y be due t o a n i n a b i l i t y of t h e p h o s p h o l i p a s e t o r e a c h t h e site 1 p. ROSENBERG and E. A. KHAIRALLAH,J. Neurochem. 23, 55 (1974).

1402

Speeialia

EXPERIENTIA 31112

Penetration of phospholipases A and C (PhA2, PhC) into axoplasm of the squid giant axon Enzyme

Enzymatic activity ~

Penetration (%) b

Type

(mg/ml)

Fresh solution

Stored incubation solution

Axoplasm

PhA~

20 20 20 1 1

7.41 6.90

5.28 6.32 4.16 0.32 0.20 0.26 0.41 0.270 0.254 0.032 0.034

18.47 13.90 14.51 1.03 0.68 0.35 0.44 0.048 0.052 0.010 0.008

0.31 0.28

I

1 PhC

10 10 1 1

0.330 0.336 0.030 0.036

350 220 349 322 340 135 107 18 20 31 24

Axoplasm was extruded following exposure of axons for 1 h to incubation solutions containing indicated concentrations of phospholipases. Incubation and axoplasm containing solutions were stored in liquid nitrogen for about 1 mouth prior to assay. 'Fresh solution' was made up within 30 rain prior to assay. Each of the 11 experiments is based upon axoplasm pooled from 4 axons. The weight of axoplasm extruded per axon ranged from 1.1 to 3.2 mg. ~PhA, [xEquiv. free fatty acids released per 10 min per p.1 PhC, increase in O.D. per 60 min per 10~zl. For purposes of calculation it is assumed that 1 mg of axoplasm equals. 1 ~xl. bActivity in axoplasm x 100 divided by activity in incubation solution.

of interest. I t t h u s is i m p o r t a n t to d e t e r m i n e w h e t h e r t h e s e e n z y m e s c a n p e n e t r a t e into t h e a x o p l a s r a of a n a x o n following e x t e r n a l application. Materials and methods. PhC, ( p h o s p h a t i d y l c h o l i n e : choline p h o s p h o h y d r o l a s e ; E C 3.1.4.3) a p a r t i a l l y p u r i f i e d p r e p a r a t i o n f r o m Clostridium per/ringens was o b t a i n e d f r o m S c h w a r z - M a n n (Orangeburg, N e w Y o r k ) ; t h e p r e p a r a t i o n (lot No. X 3452) h a s an a c t i v i t y of 15 u n i t s / m g . C o t t o n m o u t h m o c c a s i n (A gkistrodon piscivorus piscivorus ) v e n o m (lot No. 102065) w a s o b t a i n e d f r o m R o s s Allen R e p t i l e I n s t i t u t e (Silver Springs, Florida). A solution of t h i s v e n o m boiled for 15 m i n a t 100 ~ a n d p H 5 was used as t h e source of P h A 2 ( p h o s p h a f i d e acyl h y d r o l a s e ; E C 3.1.1.4) a f t e r a d j u s t m e n t of t h e s u p e r n a t a n t fluid t o p H 8.0 before use. PhA~ is t h e o n l y e n z y m e p r e s e n t in s n a k e v e n o l n s w h i c h is r e s i s t a n t t o boiling a t acid p H 2, 3. B o t h acid-boiled v e n o m a n d c o m m e r c i a l l y o b t a i n e d P h C are d e v o i d of p r o t e o l y t i c a e t i v i t y ~ - L W e c o n f i r m e d t h a t t h e s e p r e p a r a t i o n s h a d no p r o t e o l y t i c a c t i v i t y as j u d g e d b y d e t e r m i n a t i o n s s o n s u p e r n a t a n t s of TCA p r e c i p i t a t e d incubation mixtures. G i a n t a x o n s of t h e s q u i d (Loligo pealii), c o n t a i n i n g s o m e a d h e r i n g small n e r v e fibres were d i s s e c t e d as p r e v i o u s l y d e s c r i b e d 9. O n l y u n i n j u r e d a x o n s w e r e u s e d , ' t h e e n d s b e i n g carefully ligated t o a v o i d leakage of a x o p l a s m . F o r e a c h e x p e r i m e n t , 4 a x o n s ( ~ 6 0 mg) were i n c u b a t e d t o g e t h e r for 1 h, w i t h or w i t h o u t a d d i t i o n of p h o s p h o l i p a s e , in 10 m l of a n artificial sea w a t e r solution (raM c o m p o s i t i o n : NaC1, 449; KC1, 10; CaC12 50; Tris, 3; p H 8.0). T h e a x o n s were t h e n r e m o v e d f r o m t h e c h a m b e r a n d a x o p l a s m collected as d e s c r i b e d previously% 1 m l s a m p l e s of a x o p l a s m in sea w a t e r a n d t h e 10 m l of i n c u b a t i o n solutions were i m m e d i a t e l y p l a c e d in liquid n i t r o g e n u n t i l p h o s p h o l i p a s e analyses. PhA~ a c t i v i t y was d e t e r m i n e d b y t i t r a t i n g w i t h 0.02 N N a O H a c c o r d i n g t o t h e m e t h o d of DOLE 1~ t h e free f a t t y acids l i b e r a t e d a t 0, 5, 10, 15 a n d 20 rain f r o m a 1:10 dilution, in 0.9% saline, of egg yolk. T h e r e s u l t s are e x p r e s s e d as m i c r o e q u i v a l e n t s free f a t t y acid released p e r 10 rain p e r ~zl of f r e s h l y p r e p a r e d p h o s p h o l i p a s e solution, i n c u b a t i o n solution, or e x t r u d e d a x o p l a s m . P h C a c t i v i t y w a s d e t e r m i n e d b y following t h e d e v e l o p m e n t of t u r b i d i t y in egg y o l k s u s p e n s i o n s 11. A n egg y o l k is emulsified in 200 m l of 0.9% NaC1, placed in t h e r e f r i g e r a t o r for 24 h,

t h e s u p e r n a t a n t filtered a n d NaC1 a d d e d t o a final conc e n t r a t i o n of 2%. E n z y m e c o n t a i n i n g solution is a d d e d t o t h i s s u b s t r a t e (2.5 ral) a n d a b s o r b a n c e r e a d i n g s (540 nm) t a k e n e v e r y 5 rnin for 30 rain t o 3 h ; t h e increase in o p t i c a l d e n s i t y being linear w i t h t i m e . Results and discussion. A x o p l a s m f r o m a x o n s e x p o s e d t o artificial sea w a t e r alone for 1 h s h o w e d no d e t e c t a b l e level of e n d o g e n o u s P h A 2 or P h C a c t i v i t y (4 e x p e r i m e n t s ) . To d e t e r m i n e w h e t h e r p h o s p h o l i p a s e s were s t a b l e d u r i n g i n c u b a t i o n a n d storage, we c o m p a r e d t h e activities of f r e s h l y p r e p a r e d solutions of t h e s e e n z y m e s w i t h t h e a c t i v i t i e s in t h e i n c u b a t i o n solutions (Table). T h e e n z y m a t i c a c t i v i t i e s of t h e f r e s h l y p r e p a r e d a n d i n c u b a t i o n solutions w e r e similar. I n 5 e x p e r i m e n t s w i t h PhA~ t h e r e was a 3-fold a c c u m u l a t i o n of t h e e n z y m e in t h e a x o p l a s r a a n d in t h e r e m a i n i n g 2 e x p e r i m e n t s t h e e n z y m a t i c a c t i v i t i e s in t h e a x o p l a s r a a n d in t h e i n c u b a t i o n solutions were a b o u t equal. T h e P h C a c t i v i t y in t h e a x o p l a s m w a s 20 to 30% of t h a t in t h e i n c u b a t i o n solution (Table). U s i n g c o n d i t i o n s i d e n t i c a l t o t h o s e used in t h e p r e s e n t s t u d y , i t w a s f o u n d t h a t a f t e r e x p o s u r e of c o n t r o l s q u i d a x o n s t o i m p e n e t r a b l e r a d i o a c t i v e c o m p o u n d s less t h a n 1% p e n e t r a t e d into t h e a x o p l a s m a2. C o n t a m i n a t i o n is t h u s n o t a serious source of e r r o r in our studies. Our p r e s e n t results s h o w t h a t P h A 2 a n d P h C are able t o p e n e t r a t e t h r o u g h c o n n e c t i v e tissue, S c h w a n n cell a n d

2 W. L. MAGEEand R. H. S. THOMPSON,Bioehem. J. 77, 526 (1960). 8 p. ROSENBERG and K. Y. No, Biochim. biophys. Acta 75, 116 (1963). 4 H. SIMPKIIqS,S. TAY and E. PANKO,Biochemistry 10, 3579 (1971). 5 A. S. GORnON, D. F. H. WALLACHand J. H. STRAOS, Biochim. biophys. Aeta 183, 405 (1969). s D. L. MCILWAI~r and M. M. RAPPORT, Biochim. biophys. Acta 239, 71 (1971). T. K. RAy, J. DAs and G. V. MARINETTI,Chem. Phys. Lipids 9, 35 (1972). 80. H. LowRY, N. J. ROSEBROVGH,A. L. FARR and R. J. RANDALL, J. biol. Chem. 193, 265 (1951). 9 p. ROSENBERG, in Methods in Neurochernistry (Ed. R. FRIED; Marcel Dekker Inc., New York 1973), vol. 4, p. 97. zo V. P. DoL~, J. clin. Invest. 35, 150 (1956). xl. A. 0TTOLENGHI,Analyt. Biochem. 5, 37 (1963). 1~ p. ROSENBERG and F. C. G. HOSKIN,J. gen. Physiol. 46, 1065 (1963).

15.12. 1975

Specialia

a x o l e m m a , w h i c h is in a g r e e m e n t w i t h earlier d a t a w h e r e we f o u n d s p l i t t i n g of a x o p l a s m i c p h o s p h o l i p i d s following e x p o s u r e of i n t a c t s q u i d g i a n t a x o n s t o PhA~ or P h C ~, la, 14. L a c k of p e n e t r a t i o n m a y t h e r e f o r e n o t be t h e e x p l a n a t i o n for t h e f i n d i n g ~ t h a t p a n c r e a t i c P h A has no effect following e x t e r n a l a p p l i c a t i o n , b u t blocks t h e a c t i o n p o t e n t i a l of t h e s q u i d g i a n t a x o n following i n t e r n a l perfusion. U n f o r t u n a t e l y t h e y o n l y used e x t e r n a l l y one c o n c e n t r a t i o n of P h A , w h i c h was e q u a l t o t h e lowest c o n c e n t r a t i o n w h i c h h a d a n effect i n t e r n a l l y . PhA~, e x t e r n a l l y applied, causes v e s i c u l a t i o n a n d d i s r u p t i o n of t h e S c h w a n n cell of t h e s q u i d a x o n la along w i t h a large release of free a m i n o acids I i n d i c a t i v e of a b r e a k d o w n of p h o s p h o l i p i d - p r o t e i n binding, c o r r e l a t i n g well w i t h our p r e s e n t f i n d i n g s t h a t PhA~ is h i g h l y p e n e t r a b l e . The a p p a r e n t p e n e t r a t i o n of PhA~ was o f t e n a b o v e 100% w h i c h m a y i n d i c a t e a x o p l a s m i c b i n d i n g or a s s y m e t r i c p e r m e a b i l i t y p r o p e r t i e s . The h i g h p e r c e n t p e n e t r a t i o n of PhA~ m a y n o t o n l y be due to d i r e c t disr u p t i o n of p h o s p h o l i p i d s b u t m a y be due t o t h e c o n c o m i t a n t p r o d u c t i o n of l y s o p h o s p h a t i d e s , w h i c h h a v e a d e t e r g e n t a c t i o n a n d are r e s p o n s i b l e for block of c o n d u c t i o n following PhA~ a p p l i c a t i o n to t h e s q u i d g i a n t a x o n ~. I t is difficult to e x p l a i n t h e a p p a r e n t d i s c r e p a n c y bet w e e n our f i n d i n g t h a t P h C does n o t block s q u i d axoll c o n d u c t i o n following e x t e r n a l application~a,l~ e v e n t h o u g h we n o w k n o w t h a t it p e n e t r a t e s into t h e a x o p l a s m , a n d t h e finding t h a t i n t e r n a l i n j e c t i o n (1.5 ~1 of a 0.5 rag/ ml solution) or p e r f u s i o n (5 mg/ml) of p h o s p h o l i p a s e C into t h e a x o p l a s m causes a loss of e x c i t a b i l i t y in 20 to 30 m i n is (I. TASAKI, 1972, p e r s o n a l c o m m u n i c a t i o n ) .

1403

Summary. Following 1 h exposure, t h e level of p h o s p h o lipase A2 p e n e t r a t i o n i n t o t h e a x o p l a s m of t h e s q u i d g i a n t axoll was 107 t o 350% of t h a t . i n t h e e x t e r n a l m e d i a ; c o r r e s p o n d i n g values for p h o s p h o l i p a s e C were 18 t o 31%. P h o s p h o l i p a s e s Call t h e r e f o r e be used to s t u d y p h o s p h o lipid f u n c t i o n in a x o n s since t h e y can p e n e t r a t e t h r o u g h c o n n e c t i v e tissue a n d S c h w a n n cell t o r e a c h t h e a x o l e m ma. P . R O S E N B E R G 1~

Section o/ Pharmacology and Toxicology, University o~ Connecticut, School o/ Pharmacy, Storrs (Connecticut 06268, USA), 23 May 7975. 18 p. ROSENBERG, Toxicon 8, 235 (1970). 14 E. CONDREA and P. ROSENBERG, Biochim. biophys. Acta 750, 271 (1968). 15 N. J. ABBOTT, T. DEGUCIII, D. T. FRAZIER, K. ~{URAYAMA, T.

NARAHASHI, A. OTTOLENGHIand C. ~VANGJ. Physiol., Lond. 220, 73 (1972). 1~ R. MARTI~ and P. ROSENBERG J. Cell Biol. 36, 341 (1968). i7 p. ROSENBERG and E. CONDREA, Biocheln. Pharmac. 77, 2033 (1968). 18 I. TASAKIand T. TAKENAKA~ Proc. n.atn. Acad. Sci., USA 52, 804 (1964). ~ I thank Dr. TOSHIONARAHASHIfor allowing me to use his laboratory facilities at the Marine Biological Laboratory, Woods Hole, Mass. for the dissection of squid axons. Expert technical assistance in these studies were provided by Mr. STEVKN TRUDEL and Mr. STUART OWEN ROSENBERO.This work was supported in part by a grant from the University of Connecticut Research Foundation.

Evolution et contrSle de l'activit6 de l'argininosuccinate synth6tase, de l'argininosuccinase et de l'arginase du foie foetal de rat Control Activities and Regulation by the Glucocorticoids of Three Urea-Cycle E n z y m e s in Rat Foetal Liver: Argininosuccinate Synthetase, Argininosuccinase and Arginase Au cours des derniers jours de la vie foetale, de n o m breuses e n z y m e s s ' a c c u m u l e n t d a n s le foie et il est s o u v e n t possible de d6celer tr6s t 6 t leur activit6, De r & e n t e s r e c h e r c h e s o n t m o n t r 6 que la r6gulation de ces e n z y m e s ob6it, bien a v a n t la llaissance, ~ un d & e r m i n i s m e e n d o crinien. Ainsi, les t r a v a u x de J o s ' r et JAcQuo'r 1-3, de GREENGARD 4, de HOLT e t OLIVER 5, de RKIHK e t S u m KONEN 6, o n t r6v616 le r61e i m p o r t a n t que j o u e n t les glucocorticoides, le glucagon et d ' a u t r e s h o r m o n e s sur l'activit6 e n z y m a t i q u e de diverses voies m & a b o l i q u e s . Au cours d ' u n e p r & 6 d e n t e n o t e 7, les activit6s des d e u x p r e m i b r e s e n z y m e s du cycle de l'ur6e, la c a r b a m y l p h o s p h a t a s e s y n t h & a s e , (E.C. 2, 7, 2, 2) e t l ' o r n i t h i n e t r a n s c a r b a m y l a s e (E.C. 2, 1, 3, 3) ollt 4t6 mesur6es chez le foetus e t leur c o n t r h l e p a r les glucocorticoides a 4t4 mis en 6vidence. Le p r 6 s e n t t r a v a i l r a p p o r t e les r 4 s u l t a t s o b t e n u s en 6 t u d i a n t , d a n s les m ~ m e s c o n d i t i o n s exp6rim e n t a l e s que p r 6 c 6 d e m m e n t v, trois a u t r e s e n z y m e s impliqu6es d a n s la s y n t h 6 s e de F u r & , l ' a r g i n i n o s u c c i n a t e s y n t h 6 t a s e (E.C. 6, 3, 4, 5), l ' a r g i n i n o s u c c i n a s e (E.C. 4, 3, 2, 1) et l ' a r g i n a s e (E.C. 3, 5, 3, 1). Matdriels et rndthodes. L ' 6 t u d e p o r t e sur des r a t s b l a n c s S h e r m a n . L'~ge du foetus est d6termin6 p a r r a p p o r t au m o m e n t suppos6 de la f 6 c o n d a t i o n (1 h e u r e du m a t i n ) . L a s u r r 6 n a l e c t o m i e de la m6re est faite ~, 14,5 jours. Les a n i m a u x op6r6s r e f o i v e n t ~ volont6 de l ' e a u sal6e (NaC1 9 g 1 1). L ' h y p o p h y s e c t o m i e des foetus est Iaite d a n s les c o n d i t i o n s habituelles, p a r d 6 c a p i t a t i o n in u t e r o s, 9 ~ 18,5 j ours. Certains foetus d6capit4s r e g o i v e n t

a u s s i t 6 t apr6s l ' o p 6 r a t i o n 0,1 m g d ' a c & a t e de cortisol (Roussel) p a r voie i n t r a p 6 r i t o n 6 a l e au t r a v e r s du muscle ut6rin. D ' a u t r e s foetus entiers p r o v e n a n t de m6res n o n s u r r 4 n a l e c t o m i s & s re~oivent aussi c e t t e p r 6 p a r a t i o n h o r m o n a l e au m~me age (18,5 jours) et des foetus t 6 m o i n s de l ' a u t r e corne, u n v o l u m e 6 q u i v a l e n t de NaC1 ~ 9 g 1-L Au static de g e s t a t i o n 5. &udier, la m6re est a s s o m m & e t les foetus s o n t retir6s de l ' u t & u s e t saign6s. Les foies s o n t r a p i d e m e n t pr616v6s e t homog6n4is6s d a n s du b r o m u r e de N. c6tyl-N, N, N, t r i m 6 t h y l a m m o n i u m ~ 0 , 1 % p o u r la m e s u r e de l'activit6 de l ' a r g i n i n o s u c c i n a t e s y n t h & a s e e t de l ' a r g i n i n o s u c c i n a s e e t d a n s un milieu constitu6 de 9 g 1-1 de NaC1 et de 6 g 1-1 de M n S o 4, 1 H~O p o u r la m e s u r e de l'activit6 de l'arginase. Les activit6s e n z y m a t i q u e s s o n t mesur6es in v i t r o selon I~ATNER 10 p o u r

I A. JosT et R. JAcQuoL C. r. Acad. Sci., Paris 239, 98 (1954). R. JACQt:OT, C. r. Soc. Biol., Paris 150, 2137 (1956). a R. JAcQuoT, J. Physiol., Paris 51,655 (1959). 40. GREENDARD,M. K. SAHIB and W. E. KNox, Arch. Biochem. Biophys. 737, 2, 497 (1970): 5 p. G. HOLT and I. T. OLIVER, Biochemistry 8, 4 (1969). o N. C. R, R~m;i and J. SUmKONEN, Biochem. J. 107, 793 (1968). C. GAUTIER, A. HusSON et R. VAILLANT, Experientia 31, 125 (1975).

s A. A. 10 S. N.

JosT, C. r. Acad. Sci., Paris 225, 322 (1947). JOST, Arch. Anat. Microse. Morph. exp. 40, 247 (1951). RATNER, in Methods in EnzymoIogy (Eds. S. P. COLOWICKet O. KAPLAN,Acaderlfic Press, New York 1955), vol. 2, p. 356.