Intensification of the cellular accumulation of amino acids by pyridoxal

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I N T E N S I F I C A T I O N OF T H E C E L L U L A R ACCUMULATION OF AMINO ACIDS BY P Y R I D O X A L * by T H O M A S R. R I G G S , B A R B A R A

C O Y N E AND H A L V O R N. C H R I S T E N S E N

Departme~t o/Biochemistry and Nutrition, Tu/ts College ,Medical School, Boston (U.S.~4.)

I n a search for factors w h i c h m i g h t f o r m a p a r t of t h e a p p a r a t u s b y w h i c h a m i n o acids are t r a n s f e r r e d into cells a g a i n s t c o n c e n t r a t i o n gradients, p y r i d o x a l h a s been t e s t e d on t h e E h r l i c h m o u s e ascites c a r c i n o m a cell. I n spite of t h e very strong concentrative activity which t h e s e cells a l r e a d y show, t h e a d d i t i o n of D p y r i d o x a l at a p p r o p r i a t e levels increased b y 65 to 75 % t h e e x t e n t to w h i c h t h e y a c c u n m l a t e d glycine (Fig. i). A t h i g h e r levels p y r i d o x a l b e c a m e inhibitory. T h e s t i m u l a t i n g effect h a s b e e n o b t a i n e d alm o s t e q u a l l y w i t h cells f r o m mice on s t o c k diets, a n d on p y r i d o x i n e - d e f i c i e n t a n d pyridoxine-fortified diets. E v e n p r e i n c u b a t i o n of t h e ascitic fluid-cell s u s p e n s i o n for a n h o u r in t h e presence of 0. 5 millim o l a r p y r i d o x a l did n o t abolish a subseoj / " ° . q u e n t s t i m u l a t i n g effect of p y r i d o x a l w h e n t h e ascitic p l a s m a w a s replaced b y K r e b s ' R i n g e r - b i c a r b o n a t e solution. App a r e n t l y w h a t p r o d u c e s t h e s t i m u l a t i o n is a definite level of t h e p y r i d o x a l in t h e e x t r a c e l l u l a r phase. A molecule-for-molecule e n t r a n c e of p y r i d o x a l a n d a m i n o acid into t h e cell t o g e t h e r is n o t t h e ~,0 t I e x p l a n a t i o n , since w h e n glycine w a s a d d e d OOl a t a 25 millimolar level t h e n u m b e r of Pyeiao~t mMItltee ftogotltttr~¢scale; m i c r o m o l e s of e x t r a glycine e n t e r i n g t h e cells w a s five t i m e s as g r e a t as t h e t o t a l Fig. I. Effect of a d d e d p y r i d o x a l u p o n t h e a c c u m u l a q u a n t i t y of p y r i d o x a l added. tion of glycine b y t h e t u m o r cell. Glycine added, 2 P y r i d o x a l p h o s p h a t e w a s if a n y t h i n g m i c r o m o l e s p e r m l of s u s p e n s i o n . I n c u b a t i o n time, one less effective t h a n p y r i d o x a l in t h e a b o v e hour. T h e d i s t r i b u t i o n ratio is t h e ratio of t h e cellular respect. No s t i m u l a t i n g effect of p y r i d o x glycine level to t h e e x t r a c e l l u l a r level, each e x p r e s s e d a m i n e , p y r i d o x i n e , riboflavin, t h i a m i n e , in miUimoles p e r k i l o g r a m of water. T h e a v e r a g e v a l u e niacin or p a n t o t h e n i c acid could be detectfor t h e controls w a s 12.5. R e l a t i v e d i s t r i b u t i o n ratio = ed. T h e c o n c e n t r a t i o n of L-a, ~,-diaminoioo × (distribution ratio in t h e presence of a d d e d b u t y r i c acid b y t h e cells w a s also s t i m u p y r i d o x a l / d i s t r i b u t i o n ratio in t h e a b s e n c e of a d d e d lated. pyridoxal). METHODS

T h e cells f r o m 2 m l of ascitic fluid, freshly collected, were r e s u s p e n d e d in 2 ml of K r e b s ' R i n g e r b i c a r b o n a t e solution c o n t a i n i n g pyridoxal, a n d also glycine a t t w o to 3o millimolar c o n c e n t r a t i o n (two millimolar in t h e e x p e r i m e n t s of Fig. i). T h e c o n d i t i o n s of i n c u b a t i o n , c e n t r i f u g a t i o n a n d a n a l y s i s h a v e a l r e a d y been described 1. DISCUSSION

I n its c o e n z y m e f u n c t i o n p y r i d o x a l p h o s p h a t e is considered to b e c o m e linked to t h e a m i n o group of t h e a m i n o acid, p e r h a p s to f o r m a n N - p y r i d o x y l i d e n e derivative. T h e s u b s e q u e n t reaction * T h i s i n v e s t i g a t i o n h a s been s u p p o r t e d in p a r t b y a g r a n t (No. C-I268) f r o m t h e N a t i o n a l Cancer I n s t i t u t e , N a t i o n a l I n s t i t u t e s of H e a l t h , U n i t e d S t a t e s Public H e a l t h Service.

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VOL. l l

(1953)

in a few cases occurs at a fairly r e m o t e p o i n t in the molecule ~-a. Therefore it seems likely t h a t t h e formation of such derivatives m a y be a fairly general w a y b y which amino acids are " g r a s p e d " , p e r h a p s for translocation as well as for s t r u c t u r a l modification. A p p a r e n t l y it is the s i m u l t a n e o u s presence of the a m i n o acid and a definite level of pyridoxal in the extracellular fluid, r a t h e r t h a n an a d e q u a t e cellular s u p p l y of pyridoxal, which brings a b o u t the stimulation. One of the r e q u i r e m e n t s of the " c a r r i e r " hyl)othesis of active t r a n s p o r t (see for ~Xallil)[e ]{OSgNklb;R(; Ij} iS t h a t the carrier must I~: ul;,intaitwd at a hi.~her cq,ltCelltr~ttioll iJt ttne IJlltel than at the innt:'r limit ,,1" tht ~ Ilit!lllbl'~illt~ }lhilsc.

REFERENCES 1 H. N. CHRISTENSEN AND T. R. RinGs, J, Biol. Chem., 194 (1952) 57. 2 W. W. UMBREIT, W. A. W o o d AND I. C. GUNSALUS, J. Biol. Chem., 165 (1946) 731. 3 C. E. DALGLIESH, W. E. KNOX AND A. NEUBERGER, Nature, 168 (195 o) 20. 4 F. BINKLEY, G. M. CHRISTENSEN AND W. N. JENSEN, J. Biol. Chem., 194 (1952) lO9. 5 E. V. GORYACHENKOVA,Doklady Akad. Nauk. S.S.S.R., 85 (1952) 603. T. ROSENBERG, ,4cta Chem. Scan&, 2 (1948) 14. Received April I 5 t h , 1953

BIOSYNTHESIS OF MILK PROTEINS IN T H E FASTING GOAT by B. A. A S K O N A S AND P. N. C A M P B E L L

National lnstitule /or Medical Research, M i l l Hill, London, N . W . 7 (England)

E s t i m a t i o n s of the a m o u n t of a m i n o acid taken up b y the m a m m a r y gland based on arteriov e n o u s differences have suggested t h a t the free amino acids of the blood could not be the m a j o r precursors of the milk proteins since t h e y accounted for less t h a n 4 o % of the milk protein (SHAW AND PETERSON1). According to GRAHAM et al3 milk p r o t e i n nitrogen is chiefly derived from blood globulin, and REINEKE et al. a observed t h a t the m a m m a r y gland of lactating goats r e m o v e d a glycoprotein fraction from the blood. Recent studies with i n t r a v e n o u s l y injected labelled a m i n o acids. however, have s h o w n t h a t in r a b b i t (CAMPBELL AND WORK S) as well as in goat (BARRY5, WORK et al. 6, ASKONAS et al. 7) the isolated w h e y and casein proteins showed higher radioactivity t h a n the p l a s m a proteins. This suggested t h a t the free amino acids of the blood represented the m a j o r p r e c u r s o r s of the milk proteins. The a p p a r e n t contradiction between these two sets of results m a y be a t t r i b u t e d to the difficulty of determining the blood flow and small differences in the amino-acid levels in arterio-venous balance e x p e r i m e n t s (FOLLI~YS). REINEKE et al. 3 claimed from arterio-venous balance experiments t h a t in the fasting goat there was no u p t a k e of a m i n o acids b y the m a m m a r y gland in spite of the continued secretion of milk. This has been t a k e n to indicate t h a t the blood a m i n o acids are not essential for the s y n t h e s i s of milk proteins. If this were true it would indicate t h a t the m e c h a n i s m for synthesis of milk p r o t e i n s in the fasting animal was different from t h a t in the n o r m a l animal. I n order to test this possibility the following e x p e r i m e n t s were carried out with a lactating goat. The goat was given a p p r o x i m a t e l y 5 °/*c. of aSS-methionine (6 ing) b y i n t r a v e n o u s injection immediately after milking dry, and the animal was milked at frequent intervals during the following 6 h. The milk proteins were fractionated into casein and wiley protein fractions (CAMPBELL AND WORK S) and the activity of the fractions determined. The e x p e r i m e n t was t h e n repeated on the same goat after it had been starved for 48 h prior to injection of the methionine. Posterior p i t u i t a r y e x t r a c t was used to ensure complete drainage of the gland at the last milking. The activities of the milk proteins from the two e x p e r i m e n t s are c o m p a r e d in the table. I n both the n o r m a l and the fasting animal the a c t i v i t y of the milk proteins far exceeds t h a t of the p l a s m a protein, suggesting t h a t in b o t h cases the m e c h a n i s m of synthesis was the same and therefore t h a t the blood proteins are n o t tim m a j o r precursors of the milk protein. I t will be observed t h a t whereas in the control animal the radioactivity of the milk proteins reached its m a x i m u m value 13~ hours after tile injection of the a~S-methionine, in the fasting goat m a x i m a l activity was longer delayed