Amino Acid Transport in Bacteria - Advances in Chemistry (ACS

8

Amino Acid Transport in Bacteria

Effect of Nutritional and Physiological Factors

Downloaded by UCSF LIB CKM RSCS MGMT on November 23, 2014 | http://pubs.acs.org Publication Date: January 1, 1964 | doi: 10.1021/ba-1964-0044.ch008

JOSEPH T. HOLDEN Department of Biochemistry, Medical Research Institute, City of Hope Medical Center, Duarte, Calif.

T h e uptake and a c c u m u l a t i o n of v a r i o u s a m i n o a c i d s i n L a c t o b a c i l l u s a r a b i n o s u s have been d e s c r i b e d . D e f i c i e n c i e s of v i t a m i n B , b i o t i n , and pantothenic a c i d m a r k e d l y a l t e r the o p e r ation of these t r a n s p o r t s y s t e m s . A c c u m u l a t i o n c a p a c i t y i s d e c r e a s e d m o s t s e v e r e l y by a v i t a m i n B d e f i c i e n c y . T h i s effect a p p e a r s to arise indirectly from the synthesis of abnorm a l c e l l w a l l w h i c h r e n d e r s the t r a n s p o r t s y s t e m s u n u s u a l l y s e n s i t i v e to o s m o t i c f a c t o r s . K i n e t i c and o s m o t i c e x p e r i m e n t s a l s o exclude b i o t i n and pantothenate f r o m d i r e c t c a t a l y t i c i n v o l v e m e n t in the t r a n s p o r t p r o c e s s . L i k e v i t a m i n B , they affect uptake i n d i r e c t l y , p r o b a b l y through the m e t a b o l i s m of a s t r u c t u r a l cell component. T h e evidence p r e s e n t e d supports a concept of p o o l f o r m a tion in which free amino acids accumulate i n the cell through the i n t e r v e n t i o n of m e m b r a n e -localized transport catalysts. 6

6

6

Bacteria have been shown to accumulate amino acids against large app a r e n t c o n c e n t r a t i o n g r a d i e n t s , u t i l i z i n g s y s t e m s w h i c h function m a x i m a l l y when coupled to e n e r g y - p r o d u c i n g r e a c t i o n s ; H o l d e n (22) g i v e s a d e t a i l e d s u m m a r y . It i s s t i l l not p o s s i b l e to d e s c r i b e any p a r t of t h i s p r o c e s s i n m o l e c u l a r t e r m s . T h e r e i s s o m e evidence that p o o l s of a c c u m u l a t e d a m i n o a c i d s e x i s t i n an o s m o t i c a l l y a c t i v e and, p r e s u m a b l y , unbound f o r m (2,8,22), thereby i m p l y i n g that uptake o c c u r s by a m e m brane-localized active transport p r o c e s s . A m o r e detailed discussion of the r e a c t i o n m e c h a n i s m s t i l l r e q u i r e s s p e c u l a t i o n l a r g e l y u n s u p p o r t e d by evidence d e r i v e d f r o m studies on a m i n o a c i d t r a n s p o r t . I n -

9*

In Amino Acids and Serum Proteins; Stekol, J.; Advances in Chemistry; American Chemical Society: Washington, DC, 1964.


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97

deed, even these vague ' ' h y p o t h e s e s ' ' r e l y h e a v i l y on findings obtained w i t h i n o r g a n i c i o n and c a r b o h y d r a t e t r a n s p o r t s y s t e m s . T h i s paper s u m m a r i z e s an extensive effort to modify the a c t i v i t y of a m i n o a c i d t r a n s p o r t s y s t e m s b y r e n d e r i n g b a c t e r i a d e f i c i e n t i n v a r i o u s Β c o m p l e x v i t a m i n s . It w a s hoped that t h i s a p p r o a c h m i g h t p r o v i d e s o m e c l u e s to the nature of the c a t a l y s t s i n v o l v e d i n t r a n s p o r t e i t h e r because the v i t a m i n cofactor i s i n v o l v e d d i r e c t l y i n t h i s p r o c e s s o r i n d i r e c t l y because the v i t a m i n m i g h t be c o n c e r n e d i n the a v a i l a b i l i t y of s u c h a c a t a l y s t . W e w e r e g r e a t l y influenced i n t h i s d i r e c t i o n by the p r o v o c a t i v e findings of C h r i s t e n s e n and c o w o r k e r s (8, 10), who showed that the amount of a m i n o a c i d a c c u m u l a t e d by E h r l i c h a s c i t e s t u m o r c e l l s was g r e a t l y s t i m u l a t e d by p y r i d o x a l . T h i s l e d to the p r o p o s a l that the v i t a m i n s e r v e s as a c a r r i e r f o r a m i n o a c i d s d u r i n g e n t r y through the c e l l m e m b r a n e . A l t h o u g h the studies d e s c r i b e d h e r e r e v e a l e d a n u m b e r of i n s t a n c e s i n w h i c h v i t a m i n d e f i c i e n c i e s modify the c o u r s e of a m i n o a c i d a c c u m u l a t i o n , none of the effects s e e m s to a r i s e f r o m the d i r e c t p a r t i c i p a t i o n of the v i t a m i n i n the t r a n s p o r t p r o c e s s . G e n e r a l C h a r a c t e r i s t i c s of A m i n o A c i d T r a n s p o r t i n L a c t o b a c i l l u s Transport in Lactobacillus arabinosus The experiments discussed were performed with Lactobacillus a r a b i n o s u s u s i n g g l u t a m i c a c i d , a l a n i n e , and p r o l i n e as the t e s t a m i n o a c i d s . T h e d e t a i l e d e x p e r i m e n t a l p r o c e d u r e s have been d e s c r i b e d (21, 23, 27). W a s h e d r e s t i n g c e l l s a r e incubated i n a phosphate buffer w i t h the r a d i o a c t i v e a m i n o a c i d , centrifuged, and e x t r a c t e d to r e l e a s e the a m i n o a c i d , w h i c h i s then m e a s u r e d e n z y m i c a l l y o r by i s o t o p i c m e t h ods. F i g u r e 1 s u m m a r i z e s the a c c u m u l a t i o n of t h e s e a m i n o a c i d s w i t h i n L . a r a b i n o s u s d u r i n g i n c u b a t i o n i n buffer c o n t a i n i n g g l u c o s e . T h e r e i s l i t t l e o r no a c c u m u l a t i o n of the a m i n o a c i d i n the absence of a f e r m e n t able c a r b o h y d r a t e , i n d i c a t i n g an energy r e q u i r e m e n t i n the p r o c e s s . The a m i n o a c i d s can be r e l e a s e d by b r i e f e x p o s u r e of the c e l l s to b o i l i n g w a t e r , w a r m 75% ethanol, o r c o l d 5% T C A ( t r i c h l o r o a c e t i c a c i d ) . The c l o s e c o r r e s p o n d e n c e between e n z y m i c a l l y d e t e r m i n e d L - g l u t a m i c a c i d and the amount of this substance p r e d i c t e d f r o m the isotope c o n tent of c e l l e x t r a c t s shows that t h e r e i s l i t t l e m e t a b o l i c l o s s of t h i s a m i n o a c i d . T h e r e i s s o m e (5 to 10%) c o n v e r s i o n of L - to D - g l u t a m i c a c i d and s m a l l amounts of glutamine a r e f o r m e d . Radioautography r e v e a l e d no s i g n i f i c a n t c o n v e r s i o n of p r o l i n e o r alanine to c h r o m a t o graphically distinguishable metabolites. T h e l a r g e amounts of a m i n o a c i d taken up enter these c e l l s i n o p p o s i t i o n to s i z a b l e c o n c e n t r a t i o n g r a d i e n t s . A t the e x t e r n a l c o n c e n t r a tions d e s c r i b e d above ( F i g u r e 1) and a s s u m i n g an i n t r a c e l l u l a r a c c e s s i b l e w a t e r v o l u m e of 1.95 m l . p e r g r a m ( c e l l m a s s r e f e r s to d r y weight) (21), the r a t i o s of i n t e r n a l to e x t e r n a l c o n c e n t r a t i o n s a r e : g l u t a m i c a c i d , 111; a l a n i n e , 31; p r o l i n e , 26. It i s a s s u m e d that the a m i n o a c i d s a r e not bound and that they a r e u n i f o r m l y d i s t r i b u t e d throughout the a v a i l a b l e c e l l w a t e r . The e x t e r n a l c o n c e n t r a t i o n s n o r m a l l y u s e d exceed those w h i c h s a t u r a t e the i n t e r n a l c a p a c i t y . A t l o w e r l e v e l s the c o n c e n -


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ADVANCES IN CHEMISTRY SERIES


LU Ο

6 Ο Ο

Ο

5 to

-S? ο

Figure

1. Accumulation of (1) glutamic acid, β)alanine, (3) proline in L. arabinosus

and

Incubation at 37° in presence of glucose. Curves 4, 5, and 6 show relatively low accumulation of amino acids in absence of glucose. Detailed experimental procedures (27)

t r a t i o n r a t i o s a r e c o n s i d e r a b l y h i g h e r , r e a c h i n g a value of 390 f o r g l u tamic acid. The i n i t i a l r a t e of uptake a l s o i n c r e a s e s as the e x t e r n a l c o n c e n t r a tion i s r a i s e d ( F i g u r e 2). A t high c o n c e n t r a t i o n s further i n c r e m e n t s i n the e x t r a c e l l u l a r c o n c e n t r a t i o n p r o d u c e m u c h s m a l l e r i n c r e a s e s i n the a c c u m u l a t i o n r a t e , i n d i c a t i n g the i n v o l v e m e n t of a s a t u r a b l e component i n the uptake p r o c e s s . A t low c o n c e n t r a t i o n s the data c o n f o r m to the c l a s s i c a l equation of M i c h a e l i s - M e n t e n , g i v i n g a s t r a i g h t l i n e when plotted a c c o r d i n g to the L i n e w e a v e r - B u r k method. T h e r i s i n g slope of



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All values derived from isotope content of cell extracts and corrected for uptake observed at 2° in absence of glucose

the c u r v e at higher concentrations i n d i c a t e s the o p e r a t i o n of a second component w h i c h i s not saturated at l e v e l s up to 0.01 M . T h e s p e c i f i c i t y of the p r o c e s s so f a r has been studied m o s t e x t e n s i v e l y w i t h the glutamate s y s t e m u s i n g the technique of c o m p e t i t i o n . A s shown i n T a b l e I, a v a r i e t y of substances d i m i n i s h the r a t e of g l u t a mate-C uptake when p r e s e n t e d to the c e l l s s i m u l t a n e o u s l y w i t h t h i s a m i n o a c i d . G l u t a m i c - C a c i d s e r v e d as a r e f e r e n c e c o m p e t i t i v e s u b stance. G l u t a m i n e i s a c t i v e and at v e r y s h o r t incubation p e r i o d s i t s u r 1 4

1 2



100

Table I. Structural Specificity in Glutamate Accumulation


C

1 2

Competitor

None L-Glutamic acid D-Glutamic acid L-Glutamine L-Aspartic acid L-Asparagine DL-orAminoadipic acid DL-j3-Hydroxyglutamic acid DL-of-Methylglutamic acid Qf-Ketoglutaric acid Ν-Acetyl-L-glutamic acid Glutaric acid DL-Of-Aminobutyric acid DL-a-Aminovaleric acid D L - α , α'-Diaminoglutaric acid L-Glutamic acid, γ-methyl ester L-Ornithine γ-Aminobutyric acid

Relative Rate of Glutamate-C Entry 14

100 38 85 '59 50 89 81 64 59 100 90 100 92 89 100 100 80 92

14

a Uptake of L-C -glutamic acid after 5 min. at 37° from buffer containing this amino acid at 0.003M and no competitor was assigned a value of 100. Rates observed in the presence of competitor were recalculated on this basis to give the rela tive values shown. Competitive substrates were provided at 0.010M, except for racemic compounds, which were used at 0.020M.

passes glutamic acid i n competing for entry. A s p a r t i c acid also c o m petes e f f e c t i v e l y , w h e r e a s a s p a r a g i n e has l i t t l e o r no affinity f o r the a c c u m u l a t i o n c a t a l y s t . F r o m the r e l a t i v e a c t i v i t i e s of t h e s e analogs i t i s c l e a r that both the α - a m i n o and α - c a r b o x y l g r o u p s a r e r e q u i r e d f o r r e a c t i o n w i t h the a c c u m u l a t i o n c a t a l y s t . The y - c a r b o x y l group a p p e a r s a l s o to be r e q u i r e d , although a m i d e s u b s t i t u t i o n a s i n g l u t a m i n e i s an a c c e p t a b l e m o d i f i c a t i o n , s i n c e i t does not g r e a t l y r e d u c e affinity f o r the h y p o t h e t i c a l c a t a l y s t . T h e p r o c e s s shows a h i g h e r d e g r e e of s t e r e o s p e c i f i c i t y than s o m e other t r a n s p o r t s y s t e m s (4, 15, 33). B e c a u s e of the l i k e l y c o m p l e x i t y of t h i s p r o c e s s and the c o n c o m i tantly m i n o r p r o b a b i l i t y of d e r i v i n g m e c h a n i s t i c a l l y u s e f u l i n f o r m a t i o n f r o m s u c h s t u d i e s , l i t t l e attention has been g i v e n to the effects of p H on uptake beyond s h o w i n g that glutamate a c c u m u l a t i o n i s m a x i m a l between a p H of 6.0 and 6.5. T h i s i s c l o s e to the p H o p t i m u m f o r g l y c o l y s i s i n t h i s o r g a n i s m . In a g r e e m e n t w i t h the e a r l i e r findings of G a l e (14), a c c u m u l a t e d a m i n o a c i d s a r e r e t a i n e d t e n a c i o u s l y even d u r i n g w a s h i n g i n d i s t i l l e d w a t e r . T h e y c a n be eluted f r o m the c e l l s by those s u b s t a n c e s w h i c h a r e effective a s c o m p e t i t o r s d u r i n g uptake (Table I).


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Amino Acid Transport in Bacteria 1

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Γ

MINUTES Figure

3. Effect of nutritional status and growth phase on course of glutamate accumulation

time-

L. arabinosus cultured and harvested under following conditions : 1. Complete medium, cells harvested in mid-exponential phase at density of 0.35 mg./ml. 2. Complete medium, cells harvested in early exponential phase at density of 0.20 mg./ml. 3. Low biotinmedium (0.050 m^g./ml.) cells harvested in period of declining growth rate at a density of 0.15 mg./ml. 4. Low pantothenic acid medium (7.5 m\ig./ml.) cells harvested in period of declining growth rate at density of 0.23 mg./ml. 5. Low Be medium (0.020 m^g./ml.) cells harvested in period of declining growth rate at density of 0.19 mg./ml. Washed cells incubated with C -glutamic acid under standard uptake conditions (27). Glutamate uptake estimated from isotope content of cell extracts 14

Effect

of Vitamin Deficiencies

on Amino Acid

Uptake

T h e c e l l u l a r content of v a r i o u s v i t a m i n s s u c h a s n i a c i n , b i o t i n , pantothenic a c i d , and v i t a m i n B c a n be r e d u c e d to l o w l e v e l s by c u l t i 6


102


v a t i n g L . a r a b i n o s u s i n m e d i a containing t r a c e amounts of these s u b s t a n c e s . H a v i n g e x p l o r e d s o m e of the p r o p e r t i e s of the t r a n s p o r t s y s t e m s i n n u t r i t i o n a l l y n o r m a l c e l l s , this study w a s extended to an i n v e s t i g a t i o n of these s y s t e m s i n v a r i o u s v i t a m i n - d e f i c i e n t c e l l types. F i g u r e 3 s u m m a r i z e s the c o u r s e of g l u t a m i c a c i d a c c u m u l a t i o n i n some of these n u t r i t i o n a l v a r i a n t s . S i n c e these d e f i c i e n c i e s g e n e r a l l y r e d u c e the c e l l y i e l d , n u t r i t i o n a l l y n o r m a l c e l l s h a r v e s t e d at c o m p a r a b l y low d e n s i t i e s w e r e used as c o n t r o l s . T h e a c c u m u l a t i o n r a t e of a l l low density c e l l types d e c l i n e s m a r k e d l y after a b r i e f p e r i o d of n o r m a l a c c u m u l a t i o n . T h e r e i s no further a c c u m u l a t i o n by s e v e r e l y v i t a m i n B - d e f i c i e n t c e l l s . In a l l other low d e n s i t y c e l l types the a c c u m u l a t i o n r a t e s u b s e quently i n c r e a s e s w i t h l o n g e r i n c u b a t i o n . T h e onset of t h i s phase i s postponed by a b i o t i n d e f i c i e n c y , and by a pantothenic a c i d d e f i c i e n c y to an even g r e a t e r extent. B e c a u s e v i t a m i n B has been c o n s i d e r e d p r o m i n e n t l y as a potential c a r r i e r f o r a m i n o a c i d s i n c e l l u l a r t r a n s p o r t , the effects of t h i s d e f i c i e n c y w e r e i n v e s t i g a t e d i n t e n s i v e l y . A s shown i n F i g u r e 4, a v i t a m i n B d e f i c i e n c y a l s o has a pronounced effect on p r o l i n e and a l a n i n e a c c u m u l a t i o n . A l a r g e n u m b e r of e x p e r i m e n t s w i t h v i t a m i n B - d e f i c i e n t c e l l s ( L B c e l l s , grown i n absence o r p r e s e n c e of t r a c e amounts of p y r i d o x a m i n e ) showed that although t h e r e was a m a r k e d r e d u c t i o n i n t o t a l g l u t a m i c a c i d a c c u m u l a t i o n , t h e r e w a s no effect on the i n i t i a l u p take r a t e . P r e t r e a t m e n t of L B c e l l s w i t h v a r i o u s B i n h i b i t o r s s u c h as i s o n i c o t i n i c a c i d h y d r a z i d e , s e m i c a r b a z i d e , o r h y d r o x y l a m i n e d i d not d e p r e s s the i n i t i a l r a t e . T h e b a c t e r i a used i n these e x p e r i m e n t s c o n tained an a v e r a g e of only 40 m o l e c u l e s of v i t a m i n B p e r c e l l . T h e a c t i v i t i e s of v a r i o u s B - d e p e n d e n t e n z y m e s s u c h as the g l u t a m i c a c i d d e c a r b o x y l a s e , and the a s p a r t i c - g l u t a m i c t r a n s a m i n a s e w e r e d e p r e s s e d m a r k e d l y i n these c e l l s . Consequently, the attainment of a n o r m a l a c c u m u l a t i o n r a t e i s not e a s i l y r e c o n c i l e d w i t h the suggestion that the v i t a m i n functions as a c a r r i e r m o l e c u l e . If t h i s w e r e t r u e f o r t h i s b a c t e r i u m , t h e r e should have been fewer effective c a r r i e r m o l e c u l e s i n the d e f i c i e n t c e l l s and, t h e r e f o r e , a s u b s t a n t i a l l y reduced r a t e of u p take. T h e l a c k of effect of a v i t a m i n B d e f i c i e n c y on the i n i t i a l a c c u m u l a t i o n r a t e c o n t r a s t s w i t h the m a r k e d effect of t h i s d e f i c i e n c y o n the t o t a l amount of a m i n o a c i d taken up. T r e a t m e n t of deficient c e l l s w i t h v a r i o u s f o r m s of v i t a m i n B alone f a i l e d to i m p r o v e the a c c u m u l a t i o n c a p a c i t y . S i m i l a r l y , v i t a m i n B antagonists f a i l e d to reduce the c a p a c i t y of n u t r i t i o n a l l y n o r m a l c e l l s . B o t h o b s e r v a t i o n s suggested that the v i t a m i n affects a c c u m u l a t i o n c a p a c i t y i n d i r e c t l y and not by d i r e c t i n t e r v e n t i o n of a v i t a m i n B - d e p e n d e n t c a t a l y s t i n the uptake p r o c e s s . S i m u l t a n e o u s l y , i t w a s o b s e r v e d that L B c e l l s a r e m o r p h o l o g i c a l l y a b n o r m a l , h a v i n g a s w o l l e n , bulged appearance (26), and that they r e l e a s e l a r g e quantities of 260 ι η μ - a b s o r b i n g m a t e r i a l s i n c l u d i n g n u c l e o t i d e s d u r i n g i n c u b a t i o n i n buffers (19). It b e c a m e apparent that L B c e l l s might be deficient i n c e l l w a l l substance, thus a c c o u n t i n g f o r the m o r p h o l o g i c a l change. T h e d e c l i n e i n a m i n o a c i d a c c u m u l a t i o n a c t i v i t y c o u l d be d e r i v e d i n d i r e c t l y f r o m t h i s p r i m a r y s t r u c t u r a l defect, p o s s i b l y through a s e c o n d a r y change i n the c o n f i g u r a t i o n of a m e m b r a n e localized transport catalyst.


6

6

6

6

6

6

6

6

6

6

6

6

6

6

6


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20

20

40

60

M INUTES Figure

4, Effect

of vitamin Be -deficiency in L. arabinosus alanine and proline accumulation

on

Washed cells incubated under standard uptake conditions with the C -amino acid at 0.003M. Accumulation estimated from isotope content of hot water cell extracts 1 4

In a c c o r d w i t h t h i s i n t e r p r e t a t i o n , i t w a s o b s e r v e d that h i g h c o n c e n t r a t i o n s of s u c r o s e , K C 1 , and other i o n i c and nonionic substances r e s t o r e d the a c c u m u l a t i o n c a p a c i t y of L B c e l l s to n o r m a l l e v e l s (Table Π ) . The c o m p l e t e l y o s m o t i c nature of this effect i s supported by the findings s u m m a r i z e d i n F i g u r e 5, w h i c h shows that except at v e r y h i g h l e v e l s s u c r o s e and K C 1 have p r e c i s e l y equal s t i m u l a t o r y ef fects on glutamate a c c u m u l a t i o n o v e r a w i d e range of i s o - o s m o t i c c o n c e n t r a t i o n s . In c o n t r a s t to the tenacious r e t e n t i o n of a c c u m u l a t e d a m i n o a c i d s by n u t r i t i o n a l l y n o r m a l c e l l s , L B c e l l s , w h i c h have a c c u 6

6



104

Table II. Stimulation of Glutamate Accumulation by Various Substances

0

% of Maximum Stimulation 0.3 M

0.6 M

0.9 M

NH4CI

47 32 16 50 58 46 50 70

92 66 45 74 97 85 80 85

100 72 57 74 91 76 61 71

Sorbitol Fructose Potassium acetate NaCl MgCl

10 7 22 40 71

34 13 30 51 22

0 0 9

0 0 0

0 0 0

15 I I I

I I I I

I I I I

Compound

Degree of Activity

Sucrose Glucose Galactose Lysine KC1 KBr KN0


High stimulation

3

Moderate stimulation

2

No stimulation

Glycerol Rhamnose Ribose

Inhibition

Potassium lactate Glycine KH P0 KCNS 2

a

4

48 24 lb I I

L * arabinosus (LB ) was incubated for 90 min. under standard uptake conditions modified to include test substance at stated concentration. The results were taken from a number of experiments, in all of which sucrose was used as a reference stimulatory compound. I = inhibition. 6

b

m u l a t e d a l a r g e p o o l i n the p r e s e n c e of s u c r o s e , r a p i d l y l o s e t h i s a d d i t i o n a l p o r t i o n of the p o o l when suspended i n m e d i a of s u b s t a n t i a l l y l o w e r o s m o l a l i t y . T h e a b n o r m a l a c c u m u l a t i o n s of a l a n i n e and p r o l i n e i n L B c e l l s a l s o a r e c o r r e c t e d by s u c r o s e and K C L , showing that these defects have a c o m m o n b a s i s . It s e e m e d c l e a r that s u c r o s e has a b e n e f i c i a l effect on a c c u m u l a t i o n because i t r e d u c e s w a t e r content i n s o m e p o r t i o n of the c e l l . T h i s r e g i o n of the c e l l , t h e r e f o r e , should not be penetrated b y s u c r o s e . Consequently, t h i c k s u s p e n s i o n s of c e l l s w e r e exposed to v a r i o u s s u b stances to d e t e r m i n e what p o r t i o n of the c e l l space a c c e s s i b l e to a n o n s t i m u l a t o r y substance ( g l y c e r o l ) i s not entered by a s t i m u l a t o r y substance ( s u c r o s e ) . A s shown i n T a b l e Ι Π , s u c r o s e e n t e r s n u t r i t i o n a l l y n o r m a l c e l l s o n l y to a s l i g h t l y g r e a t e r extent than d e x t r a n and i n u l i n , w h i c h a r e confined to the e x t r a c e l l u l a r c o m p a r t m e n t . The s m a l l d i f f e r e n c e s b e tween these v a l u e s m a y a r i s e f r o m the m o r e c o m p l e t e d i s t r i b u t i o n of s u c r o s e i n c e l l w a l l i n t e r s t i c e s . In c o n t r a s t , g l y c e r o l , a substance w h i c h i s known to enter many c e l l s r a p i d l y and w h i c h does not s t i m u 6



8.

HOLDEN

105


S U C R O S E M O L A L I T Y AND K C l O S M O L A L I T Y Figure

5. Comparative effectiveness of sucrose and KCl in ulating glutamate accumulation by LBe cells

stim-

To maintain equal cell concentrations at equivalent levels of sucrose and CKl, amount of these substances required to give indicated molal concentration added to usual volume (3.45 ml.) of uptake buffer containing glucose and L-C -glutamic acid. Incubation for 100 minutes at37°C. Amount of glutamate calculated from isotope content of cell extracts 14

late a c c u m u l a t i o n , p e n e t r a t e s a l l but a p p r o x i m a t e l y 28% of the p e l l e t v o l u m e . T h u s , i n n o r m a l c e l l s s u c r o s e i s excluded f r o m the w h o l e c e l l i n t e r i o r , p r o b a b l y by the p e r i p h e r a l c e l l m e m b r a n e . I n v i t a m i n B deficient c e l l s these r e l a t i o n s h i p s a r e e s s e n t i a l l y the s a m e . S u c r o s e a p p e a r s to enter a s l i g h t l y l a r g e r i n t e r n a l v o l u m e . H o w e v e r , these suspensions contain a h i g h e r p r o p o r t i o n of nonviable c e l l s w h i c h m a y be c o m p l e t e l y penetrated b y s u c r o s e . T h e r e f o r e , i t a p p e a r s l i k e l y that h e r e too s u c r o s e i s excluded f r o m m o s t o r a l l the c e l l v o l u m e l y i n g beneath the s u r f a c e m e m b r a n e and that i t s t i m u l a t e s a c c u m u l a t i o n by p r e v e n t i n g w a t e r influx to t h i s r e g i o n of the c e l l . W h i l e these r e s u l t s 6



106

Table III. Permeability of L. arabinosus to Sucrose and Glycerol

0

Volume, Ml./G. DW Test Substance

HB cells

LB cells

Dextran Inulin Sucrose C -Sucrose C -Glycerol

4.16 3.15 3.04 2.88 2.50 1.15

4.43 3.13 3.19 2.71 2.33 1.28

6

Space Measured


Total pellet Total cell Sucrose impermeable

14

14

Glycerol impermeable a

6

Approximately 100 mg. of centrifuged cells were resuspended and incubated at 37° for 15 or 45 min. in an equal volume of test substance dissolved in 0.12M phosphate buffer. Dextran (av. M.W. 60,000—90,000) was used at 100 mg./ml., inulin at 50 mg./ml., sucrose at 0.2 or 0.3M, and glycerol at 0.1M. Cells were centrifuged and supernatant analyzed as described (21). All incubations were in duplicate. Values shown are averages of 4 experiments with each cell type. Indicated spaces were obtained by subtracting volume accessible to test substance from total pellet volume.

a r e c o n s i s t e n t w i t h the a c c u m u l a t i o n m o d e l w h i c h a s s i g n s a p r o m inent r o l e to the c e l l m e m b r a n e , they do not exclude the p o s s i b i l i t y that p o o l s a c c u m u l a t e w i t h i n i n t r a c e l l u l a r s i t e s w h i c h might be d i s rupted by o v e r h y d r a t i o n of the c e l l i n t e r i o r . Effect of Cell Wall Synthesis

on Amino

Acid

Accumulation

A s i n d i c a t e d above, the i n t i m a t e r e l a t i o n between a c c u m u l a t i o n c a p a c i t y and e x t r a c e l l u l a r o s m o t i c a c t i v i t y suggested that v i t a m i n B deficient c e l l s might p o s s e s s w a l l s l a c k i n g n o r m a l r i g i d i t y . T h i s i n f e r e n c e has been substantiated i n a n u m b e r of w a y s . M o s t r e c e n t l y , the r e l a t i v e d e f i c i e n c y of w a l l substance i n L B c e l l s has been d e m o n s t r a t e d d i r e c t l y by e l e c t r o n m i c r o s c o p y (22) and by d i r e c t i s o l a t i o n (25). In a c o m p a r a t i v e study u s i n g v a r i o u s n u t r i t i o n a l and p h y s i o l o g i c a l c e l l types, L B c e l l s y i e l d e d only 60% as m u c h i s o l a t a b l e w a l l substance as d i d n u t r i t i o n a l l y n o r m a l c e l l s h a r v e s t e d at a c o m p a r a b l e d e n s i t y and g r o w t h phase. M o s t of t h i s change r e p r e s e n t s a pronounced d e c r e a s e i n the amount of mucopeptide. The t e i c h o i c a c i d f r a c t i o n w a s affected m u c h l e s s , suggesting that these m a j o r w a l l p o l y m e r s m a y be s y n t h e s i z e d independently of one another. E a r l i e r i n v e s t i g a t i o n of e n v i r o n m e n t a l f a c t o r s w h i c h modify the a c c u m u l a t i o n c a p a c i t y of L B c e l l s foreshadowed m u c h of these f i n d i n g s . S p e c i f i c a l l y , i t was o b s e r v e d that conditions w h i c h favor c e l l w a l l biosynthesis promote a large increase i n amino acid accumulation c a p a c i t y . T h e s e o b s e r v a t i o n s o r i g i n a t e d i n the i n i t i a l studies on o s m o t i c p r o t e c t i o n when i t was o b s e r v e d that L B c e l l s w a s h e d w i t h v i t a m i n B 6

6

6

6

6


6

8.

HOLDEN

107


supplemented g r o w t h m e d i u m a c c u m u l a t e d e s s e n t i a l l y n o r m a l amounts of g l u t a m i c a c i d , apparently b e c a u s e a s m a l l amount of g r o w t h m e d i u m was unavoidably c a r r i e d w i t h the c e l l s into the uptake buffer (20). T h e s e s m a l l amounts of m e d i u m d i d not p r o v i d e sufficient nutrients to support s i g n i f i c a n t c e l l d i v i s i o n , and, a s shown i n T a b l e I V , the effect of g r o w t h

Table IV. Stimulation of Glutamate Accumulation


by Growth Medium Components

Glutamate Uptake, μπιοΙββ/ΙΟΟ Mg./90 Min.

Additions to Uptake Buffer

20.0 19.6

None Pyridoxamine Growth medium (+ B ) (-B )

50.4 31.2

a

6

6

Growth medium components b Vitamin mix Purines, pyrimidines + B Amino acids + B Buffer, trace salts + B

20.3 23.2 20.0 54.0

6

6

6

Buffer-salts components k NH C1, potassium acetate, B MnSQ , M g C l , K H P 0 , B 4

4

2

2

4

6

6

54.7 20.0

a

0.06 ml. used, volume calculated to be slightly in excess of that carried over when a cell pellet was washed with growth medium [cf. (28) for growth medium composition]. h At levels equal to those contained in 0.06 ml. of medium.

m e d i u m c o u l d be r e p r o d u c e d e n t i r e l y by i t s buffer and t r a c e element f r a c t i o n . T h e effect of these components i n t u r n w a s accounted f o r e n t i r e l y by acetate, N H , and p y r i d o x a m i n e . U n d e r these conditions n e a r l y n o r m a l a c c u m u l a t i o n c a p a c i t y w a s a c h i e v e d i n the absence of o s m o t i c p r o t e c t a n t s . T h e s e b e n e f i c i a l changes w e r e o b s e r v e d i n the absence of s i g n i f i c a n t i n c r e a s e s i n i n t r a c e l l u l a r p r o t e i n and n u c l e i c acid. T h e effect of acetate i s h i g h l y s p e c i f i c . Of a l a r g e v a r i e t y of m o n o - and d i c a r b o x y l i c a c i d s tested, only a few s u c h as p y r u v a t e , b u t y r a t e , m a l a t e , and succinate s t i m u l a t e d uptake s l i g h t l y even when tested at concentrations 5 t i m e s h i g h e r than those at w h i c h acetate i s effective. P y r i d o x a m i n e w a s m a x i m a l l y effective at v e r y low l e v e l s (1 x 10 " M ) , i n d i c a t i n g a c a t a l y t i c r o l e i n t h i s phenomenon. In the range w h e r e i t s c o n c e n t r a t i o n i s l i m i t i n g , the t o t a l amount of N H a v a i l a b l e w a s l o w e r than the a d d i t i o n a l glutamate a c c u m u l a t e d . A c l e a r e r i n d i c a t i o n of the s t i m u l a t o r y m e c h a n i s m c a m e f r o m p r e t r e a t m e n t studies. T h e s e w e r e attempted on the p r e m i s e that i f these substances a r e u s e d to r e p a i r a c e l l w a l l defect, i t should be p o s s i b l e +

4

9

+

4



108 +

to t r e a t c e l l s w i t h acetate, N H , and v i t a m i n B and thereby b r i n g t h e m to a state i n w h i c h they w o u l d a c c u m u l a t e a m i n o a c i d n o r m a l l y i n the absence of these s t i m u l a t o r y compounds o r o s m o t i c p r o t e c t a n t s . In s u c h e x p e r i m e n t s L B c e l l s w e r e p r e t r e a t e d i n s u c r o s e buffer w i t h a c e tate, N H , and p y r i d o x a m i n e , centrifuged away f r o m these substances, and then exposed to g l u t a m i c - C a c i d under standard uptake conditions to m e a s u r e t h e i r a c c u m u l a t i o n c a p a c i t y . I n i t i a l l y , t h i s p r o c e d u r e f a i l e d to p r o d u c e any i m p r o v e m e n t i n a m i n o a c i d a c c u m u l a t i o n . H o w e v e r , as shown i n T a b l e V , the a d d i t i o n of g l u t a m a t e - C to the p r e t r e a t m e n t s o 4

6

6

+

4

1 4


12

Table V. Effect of Preincubation on Restoration of Glutamate Accumulation Activity in L B C e l l s 0

e

14

Time, min.

Uptake Condition

Glutamate (C ) Accumulation, μ moles/100 Mg./90 Min.

0 0 90 90 90

Stda + Mix Std Std Std

9.9 52.6 13.6 13.4 46.3

Preincubation Supplement Sucrose

+ mix a + mix + L-glutamate (1 M)

a

L B cells of L. arabinosus were preincubated under standard uptake conditions (27) with indicated supplements. Sucrose was used at 0.6M. Mix provides po tassium acetate at a final concentration of 0.0023M, NH C1 at 0.0012M, and pyridoxamine at 0.18 χ 10~ M. After times shown cells were separated from this solution by centrifugation at 5000 x g for 7 min., resuspended in cold buf fer, and distributed to tubes for measurement of C -glutamate accumulation in normal way. Std. refers to standard uptake condition. 6

4

6

14

l u t i o n c a u s e d a l a r g e i n c r e a s e i n the subsequently m e a s u r e d a c c u m u lation activity. W h i l e t h i s suggests that glutamate i s an e s s e n t i a l component of the p r e t r e a t m e n t s o l u t i o n , an a l t e r n a t e p o s s i b i l i t y i s that the c e l l m u s t a c c u m u l a t e glutamate to p r o t e c t o r m a i n t a i n an e s s e n t i a l i n t r a c e l l u l a r s i t e . A n u m b e r of o b s e r v a t i o n s c o n f l i c t w i t h the l a t t e r p o s s i b i l i t y . The amount of glutamate taken up and r e t a i n e d t h r o u g h the c y c l e of p r e t r e a t m e n t and w a s h i s only one half the a d d i t i o n a l amount of g l u t a m a t e C subsequently taken up. A m o r e c o n c l u s i v e f i n d i n g i s that the c a p a c ity f o r a l a n i n e a c c u m u l a t i o n i s not i n c r e a s e d when a l a n i n e i s p r o v i d e d d u r i n g p r e t r e a t m e n t , w h e r e a s t h e r e i s a s i g n i f i c a n t i m p r o v e m e n t when g l u t a m i c a c i d i s i n c l u d e d i n the p r e t r e a t m e n t s o l u t i o n . W h i l e these r e s u l t s a r e c o n s i s t e n t w i t h the p r o p o s e d i n v o l v e m e n t of the c e l l w a l l i n the d e t e r m i n a t i o n of a c c u m u l a t i o n c a p a c i t y , they do not c o m p l e t e l y e x c l u d e changes i n s o m e other c e l l component w h i c h m i g h t be the p r i m a r y l o c u s of the d e s c r i b e d effects. Consequently, the fate of a c e t a t e - C i n t h i s o r g a n i s m w a s i n v e s t i g a t e d . T a b l e V I shows that the m a j o r s i t e s of acetate i n c o r p o r a t i o n i n r e s t i n g c e l l s a r e the 1 4

14


8.

HOLDEN

109

Amino Acid Transport in Bacteria Table VI. Distribution of Acetate-2-C" in L. arabinosus Cell Fractions

Cell Type HBg

Fraction

LBg


CPM/10-Mg. Cells a Cold TCA Hot TCA Ethanol Trypsin Residue (wall)

6,000 300 128,000 500 19,000

3,000 100 114,000 1,600 38,000

awashed cells were incubated at 1.6 mg./ml. in 0.12M phosphate buffer (27) containing glucose (0.028M), sucrose (0.5M), and potassium acetate-2-C (0.0059M). After 60 min. at 37° cells were centrifuged, pellets frozen and extracted as described by Park and Hancock (39). Appropriate aliquots were plated and radioactivity was measured in a gas-flow counter. 14

e t h a n o l - s o l u b l e and the c e l l w a l l ( s p e c i f i c a l l y , mucopeptide) f r a c t i o n s . V i t a m i n B - d e f i c i e n t c e l l s i n c o r p o r a t e half as m u c h acetate into the w a l l f r a c t i o n as do n u t r i t i o n a l l y n o r m a l c e l l s , but a r e as a c t i v e as these c o n t r o l c e l l s when N H , p y r i d o x a m i n e , and L - g l u t a m i c a c i d a r e p r o v i d e d (22,24). T h e r e i s a s i z a b l e i n c r e a s e i n c e l l w a l l substance under these c o n d i t i o n s . T h u s , those p r e t r e a t m e n t conditions w h i c h f o s t e r a l a r g e i n c r e a s e i n a m i n o a c i d t r a n s p o r t and a c c u m u l a t i o n a c t i v i t y i n L B c e l l s a r e a l s o r e q u i r e d to obtain a l e v e l of acetate i n c o r p o r a t i o n into c e l l w a l l c o m p a r a b l e to that o b s e r v e d i n H B c e l l s (grown w i t h an e x c e s s of p y r i d o x a m i n e ) . Since w e have been unable to detect changes i n other c e l l f r a c t i o n s which correlate with improvements i n accumulation capacity, it app e a r s r e a s o n a b l e to conclude, e s p e c i a l l y i n v i e w of the p r e v i o u s l y c i t e d o s m o t i c evidence, that a l a c k i n c e l l w a l l r i g i d i t y l i m i t s the a c c u m u l a t i o n c a p a c i t y of L B c e l l s and that the r e p a i r of the w a l l defect suffices to p e r m i t these c e l l s to e x p r e s s n o r m a l a c c u m u l a t i o n c a p a c i t y . O n the question of the p a r t i c i p a t i o n of v i t a m i n B i n a m i n o a c i d t r a n s p o r t , these, and e s p e c i a l l y the o s m o t i c e x p e r i m e n t s , a r e c l e a r l y i n c o n s i s t e n t w i t h the suggested c a t a l y t i c r o l e of t h i s substance d i r e c t l y i n the e n t r y reaction. 6

+

4

e

6

6

6

Stimulation

of Accumulation

in Other Vitamin-Deficient

Cell

Types

T h e u n u s u a l c o u r s e of a m i n o a c i d a c c u m u l a t i o n i n b i o t i n - and p a n tothenate-deficient c e l l s ( F i g u r e 3) a l s o has been found to be m a r k e d l y influenced by s u c r o s e and other o s m o t i c p r o t e c t a n t s , a s w e l l as by a c e tate. T h e c o u r s e of g l u t a m i c a c i d uptake b y b i o t i n - d e f i c i e n t c e l l s i n the




110

MINUTES Figure

6. Effect observed

of sucrose on abnormal glutamate accumulation in biotin-deficient cells of L. arabinosus

1. Nutritionally

normal control cells, glutamate uptake measured under standard conditions 2. Biotin-deficient cells, uptake measured in presence of 0.5M sucrose 3. Biotin-deficient cells, uptake measured under standard conditions Glutamate accumulation estimated from isotope content of hot water cell extracts absence and p r e s e n c e of s u c r o s e i s d e s c r i b e d i n F i g u r e 6. T h e u n u s u a l biphasic t i m e - c o u r s e characterized by a marked decline i n a c c u m u l a t i o n r a t e after 5 m i n u t e s of n o r m a l uptake i s r e s t o r e d to the c o n t r o l p a t t e r n by high s u c r o s e c o n c e n t r a t i o n s . T h u s , as i n a v i t a m i n B d e f i 6



8.

HOLDEN


111

c i e n c y , a b i o t i n d e f i c i e n c y has no effect on the r a t e o r amount of a m i n o a c i d taken up, s o l o n g as the c e l l s a r e exposed to a h i g h e x t r a c e l l u l a r o s m o t i c p r e s s u r e . T h i s suggests, f i r s t , that these c e l l s a l s o m a y p o s s e s s s t r u c t u r a l features w h i c h a r e i n c o m p a t i b l e w i t h the m a s s i v e a c c u m u l a t i o n of a m i n o a c i d s , and, second, that t h i s v i t a m i n a l s o does not p l a y a c a t a l y t i c r o l e d i r e c t l y i n the t r a n s p o r t p r o c e s s . T h e s a m e i s t r u e f o r pantothenate-deficient c e l l s , w h i c h suffer even a m o r e p r o nounced a b e r r a t i o n i n the uptake p a t t e r n ( F i g u r e 3) but w h i c h a c c u m u late a m i n o a c i d s i n a n o r m a l m a n n e r when e x t r a c e l l u l a r s u c r o s e i s provided. W i t h b i o t i n - d e f i c i e n t c e l l s low c o n c e n t r a t i o n s of acetate w i l l s u b stitute f o r high concentrations of s u c r o s e i n r e s t o r i n g uptake to n o r m a l l e v e l s (22). B i o t i n s t i m u l a t e s s l i g h t l y when p r o v i d e d i n a d d i t i o n to a c e tate. Pantothenate-deficient c e l l s r e s p o n d d r a m a t i c a l l y to acetate only i n the p r e s e n c e of t h i s v i t a m i n . T h i s b e h a v i o r p r o b a b l y r e f l e c t s the i n v o l v e m e n t of c o e n z y m e A i n the p r o c e s s w h i c h r e s t o r e s a n o r m a l a c cumulation pattern. T h e r e f o r e , the t h r e e v i t a m i n d e f i c i e n c i e s so f a r studied i n d e t a i l appear to affect a m i n o a c i d t r a n s p o r t and a c c u m u l a t i o n i n s i m i l a r but i n d i r e c t w a y s . T h e a c c u m u l a t i o n defect i s m o s t pronounced i n v i t a m i n B - d e f i c i e n t c e l l s , f o r w h i c h t h e r e i s a l s o s t r o n g evidence i m p l i c a t i n g an a b n o r m a l i t y i n c e l l w a l l c o m p o s i t i o n as a l i k e l y s o u r c e of the change i n t r a n s p o r t a c t i v i t y . D i r e c t evidence f o r a c e l l w a l l change i n b i o t i n - and pantothenate-deficient c e l l s has not yet been obtained. T h e p o s s i b i l i t y r e m a i n s , t h e r e f o r e , that the change i n a c c u m u l a t i o n a c t i v i t y m a y be caused by an a b n o r m a l i t y i n s o m e other s t r u c t u r a l component s u c h as the p e r i p h e r a l c e l l m e m b r a n e . 6

Mechanism

of Amino Acid

Transport

and

Accumulation

T h e s e studies s t r o n g l y c o n t r a d i c t the suggestion that v i t a m i n B s e r v e s as a c a r r i e r of a m i n o a c i d s d u r i n g t r a n s p o r t , at l e a s t s o f a r as L . a r a b i n o s u s and r e l a t e d b a c t e r i a a r e c o n c e r n e d . T h e y a l s o cast s e r i ous doubt on the p o s s i b i l i t y that b i o t i n and pantothenic a c i d a r e i n v o l v e d d i r e c t l y i n this p r o c e s s . R e c e n t studies w i t h E h r l i c h a s c i t e s t u m o r c e l l s suggest that h e r e too v i t a m i n B does not c a t a l y z e the e n t r y step of t r a n s p o r t (9, 38). O n the other hand, M o r a and S n e l l (37) have o b s e r v e d that p y r i d o x a l does s t i m u l a t e a m i n o a c i d a c c u m u l a t i o n i n p r o t o p l a s t s but not i n whole c e l l s of S. f a e c a l i s . A s w i t h E h r l i c h a s c i t e s t u m o r c e l l s r e l a t i v e l y high l e v e l s of the v i t a m i n a r e r e q u i r e d to e l i c i t the r e s p o n s e . T h i s finding, together w i t h the o b s e r v a t i o n that c e l l s w i t h e x t e r n a l w a l l s do not show the effect, suggests that a s t r u c t u r a l r a t h e r than a c a t a l y t i c p r o c e s s m a y be affected. A n u m b e r of studies have shown that v i t a m i n B m a y affect a m i n o a c i d t r a n s p o r t i n i n t e s t i n a l t i s sue (31). H o w e v e r , the m e t a b o l i c b a s i s of t h i s effect has not yet been evaluated. T h e e x p e r i m e n t s r e p o r t e d h e r e a l s o have been useful i n e v a l u a t i n g v a r i o u s c u r r e n t p r o p o s a l s r e g a r d i n g the nature of the a c c u m u l a t i o n p r o c e s s . T h e v i e w i s w i d e l y h e l d that a c c u m u l a t i o n s of the type d e s c r i b e d h e r e can be a t t r i b u t e d to the o p e r a t i o n of t r a n s p o r t c a t a l y s t s 6

6

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l o c a l i z e d i n a p e r m e a b i l i t y b a r r i e r w h i c h a l l o w s net i n w a r d fluxes to be m a i n t a i n e d u n t i l h i g h i n t r a c e l l u l a r c o n c e n t r a t i o n s of f r e e a m i n o a c i d s a r e attained. T h i s t h e s i s i s supported b y the repeated d e m o n s t r a t i o n of a p e r i p h e r a l p e r m e a b i l i t y b a r r i e r i n b a c t e r i a (22, 35, 43). A l though d i r e c t efforts to d e m o n s t r a t e s e q u e s t r a t i o n of l a r g e a m i n o a c i d p o o l s i n o r on i n t r a c e l l u l a r p o l y m e r s o r p a r t i c u l a t e s have f a i l e d , and the e x t r a c t i o n p r o p e r t i e s of these p o o l s a r e c o n s i s t e n t w i t h t h e i r o c c u r r e n c e i n a f r e e f o r m , i n c o n t r o v e r t i b l e evidence s u p p o r t i n g t h i s v i e w i s s t i l l l a c k i n g . Indeed, after c a r e f u l a n a l y s i s s o m e w o r k e r s i n t e r p r e t t h e i r findings on a m i n o a c i d uptake i n E . c o l i i n t e r m s of f i x a t i o n on a d s o r p t i o n s i t e s f o l l o w i n g entrance c a t a l y z e d by m o b i l e c a r r i e r s (6,7). T h e m o s t convenient e x p e r i m e n t a l m e a s u r e of the state of the p o o l s e e m s to be i t s i n s i t u o s m o t i c a c t i v i t y . T h e r e have been a n u m b e r of attempts to d e t e r m i n e s u c h a c t i v i t y (1, 22, 36). W h i l e a m i n o a c i d s w i t h i n b a c t e r i a l p r o t o p l a s t s appear to c a u s e w a t e r influx, m e a s u r e d i n d i r e c t l y as changes i n l i g h t s c a t t e r i n g , the studies r e p o r t e d so f a r have not been q u a n t i t a t i v e l y p r e c i s e enough to e s t a b l i s h how m u c h of the p o o l p a r t i c i p a t e s i n t h i s phenomenon. A l t h o u g h they a r e a good d e a l l e s s d i r e c t , the studies r e p o r t e d h e r e a r e of i n t e r e s t f r o m t h i s point of v i e w . A n obvious i n t e r p r e t a t i o n of our findings i s that the n e w l y a c c u m u lated a m i n o a c i d p o o l i s f r e e and that an i n c r e a s e d i n t r a c e l l u l a r o s m o t i c p r e s s u r e m a y be developed i n the c o u r s e of i t s uptake. U n d e r m o s t c i r c u m s t a n c e s the c e l l w a l l i s s u f f i c i e n t l y r i g i d to p r e v e n t any a d v e r s e effects a r i s i n g f r o m the r e s u l t a n t s t r e s s . H o w e v e r , i n v i t a m i n B - d e f i c i e n t c e l l s , and p o s s i b l y i n other d e f i c i e n t types a s w e l l , w a l l r i g i d i t y i s d e c r e a s e d and the i n c r e a s i n g o s m o t i c p r e s s u r e a s s o c i a t e d w i t h a c c u m u l a t i o n c a u s e s m e m b r a n e d i s t e n t i o n o r s o m e other c o n f i g u r a t i o n a l m o d i f i c a t i o n w h i c h i s d e l e t e r i o u s to the uptake p r o c e s s . C o n sequently, a g r e a t l y d i m i n i s h e d p o o l s i z e i s attained u n l e s s the i n c r e a s e d i n t r a c e l l u l a r o s m o t i c p r e s s u r e i s offset b y a g r e a t l y elevated e x t r a c e l l u l a r osmotic p r e s s u r e . Unfortunately, this s i m p l e i n t e r p r e tation of our o s m o t i c findings i s not s t r o n g l y supported when the q u a n t i t a t i v e a s p e c t s of t h i s phenomenon a r e c o n s i d e r e d . F i g u r e 7 shows that a s the s u c r o s e c o n c e n t r a t i o n i s r a i s e d i n c r e ments i n a l a n i n e and p r o l i n e a c c u m u l a t i o n f a l l a l o n g a s t r a i g h t l i n e having the s a m e s l o p e . A l a n i n e c a p a c i t y , b e i n g h i g h e r , r e q u i r e d h i g h e r concentrations of s u c r o s e f o r m a x i m u m a c c u m u l a t i o n . H o w e v e r , a p p r o x i m a t e l y 4 μ π ι ο ΐ β β p e r m l . of e x t r a c e l l u l a r s u c r o s e w e r e r e q u i r e d to s t i m u l a t e an a d d i t i o n a l i n t r a c e l l u l a r a l a n i n e o r p r o l i n e l o a d of only 1 μ m o l e p e r m l . ( a s s u m i n g u n i f o r m d i s t r i b u t i o n of these a m i n o a c i d s i n the a v a i l a b l e c e l l w a t e r ) . If these substances have a c t i v i t y coefficients i n the v i c i n i t y of unity, i t w o u l d appear e i t h e r that other substances contribute to the o s m o t i c l o a d o r that the a m i n o a c i d s a r e s e q u e s t e r e d i n a f r a c t i o n of the c e l l w a t e r w i t h a c o r r e s p o n d i n g i n c r e a s e of c o n c e n t r a t i o n i n t h i s r e g i o n . T h e b e h a v i o r of g l u t a m i c a c i d i s m o r e e a s i l y r e c o n c i l e d w i t h the s i m p l e concept of d i r e c t o s m o t i c i n t e r p l a y of e x t r a c e l l u l a r s u c r o s e and i n t r a c e l l u l a r a m i n o a c i d . In the r a n g e 0.2 to 0 . 5 M s u c r o s e , an i n c r e a s e of 2 μ π ι ο ΐ β β p e r m l . i n e x t r a c e l l u l a r s u c r o s e c o n c e n t r a t i o n p e r m i t s an apparent i n c r e a s e of 0.84 μ π ι ο ΐ β p e r m l . of intracelluk glutamate. T h e s e v a l u e s at l e a s t a p p r o a c h the r a t i o e x 6


8.

HOLDEN

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60 h


Ο

8

ο ε

0.2

0.4

0.6

SUCROSE ( M ) Figure 7. Comparative stimulation of glutamic acid, alanine, and proline accumulation in vitamin B%-deficient cells of L. arabinosus at various extracellular sucrose concentrations Incubation at 37° for 60 minutes using indicated sucrose concentration in standard uptake buffer containing L-C amino acid at 0.003M. Accumulation calculated from iso tope content of hot water cell extracts 1 4

pected i f t h e r e w a s o s m o t i c i n t e r a c t i o n (somewhat l e s s than two a s s u m i n g that at the i n t r a c e l l u l a r p H glutamate behaves a s a u n i - u n i v a l e n t i o n of the m o n o b a s i c s a l t ) . T h u s , i n quantitative t e r m s t h i s p o r t i o n of the i n v e s t i g a t i o n g i v e s only r a t h e r l i m i t e d support to the concept of free i n t r a c e l l u l a r amino acid pools.



114

F u r t h e r support c o m e s f r o m the studies r e l a t i n g c e l l w a l l b i o s y n t h e s i s and a m i n o a c i d a c c u m u l a t i o n c a p a c i t y i n v i t a m i n B - d e f i c i e n t c e l l s , s i n c e i t i s d i f f i c u l t to account f o r these o b s e r v a t i o n s without a t t r i b u t i n g c o n s i d e r a b l e o s m o t i c a c t i v i t y to the a c c u m u l a t e d a m i n o a c i d s . A n y d e s c r i p t i o n of a c c u m u l a t i o n w h i c h i n v o k e s a m i n o a c i d attachment to i n t r a c e l l u l a r b i n d i n g s i t e s , whose affinity c a n be r e d u c e d b y a v i t a m i n B d e f i c i e n c y , m u s t account f o r the s t i m u l a t i o n of uptake that a c c o m p a n i e s the s y n t h e s i s of e s s e n t i a l l y e x t r a c e l l u l a r c e l l w a l l m a t e r i a l . If the r e d u c t i o n i n affinity o c c u r s because the c e l l i n t e r i o r b e c o m e s o v e r h y d r a t e d (a r e a s o n a b l e postulate w h i c h f o l l o w s f r o m the o s m o t i c e x p e r i m e n t s ) , the b e n e f i c i a l effect of w a l l s y n t h e s i s i s not r e a d i l y e x p l i c a b l e , s i n c e v i t a m i n B - d e f i c i e n t c e l l s have a s w o l l e n appearance w h i c h i s not s i g n i f i c a n t l y a l t e r e d after w a l l s y n t h e s i s has been s t i m u lated. T h u s , the e x i s t i n g o v e r h y d r a t i o n w i t h i n the c e l l p r o b a b l y i s not r e v e r s e d b y t h i s change. In c o n t r a s t , the d e p o s i t i o n of a d d i t i o n a l w a l l substance w o u l d p r e v e n t f u r t h e r unfavorable consequences of s w e l l i n g s u c h as m e m b r a n e d i s t e n t i o n , and, i n t h i s w a y , f o r e s t a l l the p r e m a t u r e c e s s a t i o n of a m i n o a c i d a c c u m u l a t i o n . A l t h o u g h these r e s u l t s appear to be m o s t r e a s o n a b l y accounted f o r i n t e r m s of an o s m o t i c a l l y a c t i v e p o o l w h i c h i s l a r g e l y u n a s s o c i a t e d w i t h i n t r a c e l l u l a r b i n d i n g s i t e s , the s p e c u l a t i v e nature of these p r o p o s a l s i s c l e a r l y a p p r e c i a t e d , a s i s the n e c e s s i t y f o r m o r e d e f i n i t i v e i n f o r m a t i o n . F o r e x a m p l e , w h i l e o u r evidence c l e a r l y shows an a s s o c i a t i o n between i m p r o v e m e n t s i n uptake a c t i v i t y and s y n t h e s i s of w a l l substance, i t does not exclude the c o n c o m i t a n t s y n t h e s i s of s m a l l amounts of functionally i m p o r t a n t m e m b r a n e substance. F u r t h e r m o r e , a l t e r n a t i v e p r o p o s a l s a r e c o n c e i v a b l e that w o u l d account f o r a l l our findings without n e c e s s a r i l y i n v o k i n g the o c c u r r e n c e of an o s m o t i c a l l y a c t i v e p o o l . One i s that v i t a m i n B m i g h t act as an e s s e n t i a l component of a r e g u l a t o r y m e c h a n i s m w h i c h c o n t r o l s the movement of w a t e r through the c e l l m e m b r a n e . T h e d i f f i c u l t y of e x c l u d i n g s u c h a p r o p o s a l u n d e r s c o r e s the l i m i t a t i o n s of e x i s t i n g evidence c o n c e r n i n g the m e c h a n i s m of p o o l f o r m a t i o n and r e t e n t i o n . R e c e n t studies of i n o r g a n i c i o n t r a n s p o r t have r e v e a l e d p o s s i b l e r e l a t i o n s to A T P m e t a b o l i s m (40, 42), p h o s p h o l i p i d e t u r n o v e r (18), phosphoprotein t u r n o v e r (3, 32), and o x i d a t i v e p h o s p h o r y l a t i o n (5, 41). T h e s e studies have opened f r e s h approaches to t h i s p r o b l e m and have r a i s e d hopes that some i n s i g h t into the m e c h a n i s m of i o n t r a n s p o r t w i l l soon be attained. In c o n t r a s t , p r o g r e s s i n studies on a m i n o a c i d t r a n s p o r t has been somewhat l e s s d r a m a t i c . A n u m b e r of w o r k e r s have o b s e r v e d a m i n o a c i d s i n l i p i d e e x t r a c t s , i n c l u d i n g those of m i c r o b i a l o r i g i n (11, 12, 17, 29). R e c e n t l y , M a c f a r lane (34) has r e p o r t e d that m o s t of the phospholipide i n C l o s t r i d i u m w e l c h i i i s bound to a m i n o a c i d s and that s o m e of this m a t e r i a l o c c u r s as the O - a m i n o a c i d e s t e r of p h o s p h a t i d y l g l y c e r o l . T h e r e l a t i v e l y p r o m i n e n t o c c u r r e n c e of l i p i d e s i n c e l l m e m b r a n e s has l e d to the r e c u r r e n t suggestion that t r a n s p o r t of h y d r o p h i l i c substances through s u c h m e m b r a n e s would be g r e a t l y f a c i l i t a t e d b y c o m b i n a t i o n w i t h h y d r o p h o b i c substances. Consequently, m o s t w o r k e r s who have o b s e r v e d the i n c o r p o r a t i o n of a m i n o a c i d s i n t o l i p i d e f r a c t i o n s quite n a t u r a l l y 6


6

6

6



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have mentioned the p o s s i b i l i t y that these l i p o a m i n o a c i d c o m p l e x e s a r e r e l a t e d to the t r a n s p o r t p r o c e s s (13, 16, 29, 30, 34). A s yet, t h e r e have been no d e f i n i t i v e o b s e r v a t i o n s f a v o r i n g t h i s v i e w , and s o m e i n s t a n c e s of c o n t r a r y f i n d i n g s . F o r e x a m p l e , H u n t e r and G o o d s a l l (29) have o b s e r v e d that c h l o r a m p h e n i c o l i n h i b i t s l i p o a m i n o a c i d s y n t h e s i s i n B . m e g a t e r i u m without affecting the uptake of a m i n o a c i d s into the p o o l . T h i s f i n d i n g tends to support a n a l t e r n a t i v e p r o p o s a l w h i c h has perhaps been quoted even m o r e w i d e l y , that l i p o a m i n o a c i d c o m p l e x e s a r e c o n cerned i n protein synthesis. It i s apparent that at this stage of development d e f i n i t i v e c o n c l u s i o n s a r e p r e m a t u r e , and that t h i s aspect of a m i n o a c i d and l i p i d e m e t a b o l i s m w i l l be p u r s u e d v i g o r o u s l y i n the near future. It i s of c o n s i d e r a b l e i n t e r e s t to us that b i o t i n and pantothenic a c i d d e f i c i e n c i e s affect a m i n o a c i d t r a n s p o r t i n L . a r a b i n o s u s , s i n c e both v i t a m i n s a r e known to play a prominent role i n lipide biosynthesis. We are currently r e e x a m i n i n g the t u r n o v e r of l i p i d e f r a c t i o n s i n n u t r i t i o n a l l y n o r m a l and v i t a m i n - d e f i c i e n t c e l l types to d e t e r m i n e whether there i s some r e l a tion between this aspect of m e t a b o l i s m and a m i n o a c i d t r a n s p o r t . In any c a s e , the nature of the c a t a l y t i c steps i n v o l v e d i n a m i n o a c i d t r a n s p o r t i s s t i l l unknown t o u s . T h e y p r o b a b l y o c c u r i n the p e r i p h e r a l c e l l m e m b r a n e , but even t h i s e l e m e n t a r y and w i d e l y accepted b e l i e f w i l l r e q u i r e a d d i t i o n a l study before i t can be accepted beyond doubt as an e s t a b l i s h e d fact. Summary The uptake and a c c u m u l a t i o n of v a r i o u s a m i n o a c i d s i n L a c t o b a c i l lus a r a b i n o s u s have been d e s c r i b e d . A n e x t e n s i v e i n v e s t i g a t i o n of t h i s p r o c e s s u s i n g c e l l s deficient i n v i t a m i n B , b i o t i n , and pantothenic a c i d has shown that a l l these d e f i c i e n c i e s m a r k e d l y a l t e r the t r a n s p o r t p r o c ess. Accumulation capacity i s most severely decreased by a vitamin B d e f i c i e n c y . The evidence now a v a i l a b l e i n d i c a t e s that t h i s does not r e f l e c t the d i r e c t p a r t i c i p a t i o n of the v i t a m i n i n the t r a n s p o r t p r o c e s s , but r a t h e r i s an i n d i r e c t effect a r i s i n g f r o m the s y n t h e s i s of an a b n o r m a l c e l l w a l l w h i c h r e n d e r s the c e l l u n u s u a l l y s e n s i t i v e to o s m o t i c stress. Amino acid transport i n vitamin B -deficient cells i s restored to n o r m a l l e v e l s by r a i s i n g the e x t r a c e l l u l a r o s m o t i c p r e s s u r e o r by enabling the c e l l s to s y n t h e s i z e a d d i t i o n a l w a l l substance. T h e s e findings can be i n t e r p r e t e d to support a concept of p o o l f o r m a t i o n i n w h i c h f r e e a m i n o a c i d s a c c u m u l a t e w i t h i n the c e l l through the i n t e r v e n t i o n of m e m b r a n e - l o c a l i z e d t r a n s p o r t c a t a l y s t s . T h e nature of these c a t a l y s t s i s s t i l l unknown. T h e k i n e t i c and o s m o t i c e x p e r i m e n t s r e p o r t e d h e r e a l s o appear to exclude b i o t i n and pantothenic a c i d f r o m d i r e c t i n v o l v e m e n t i n the t r a n s p o r t p r o c e s s . T h e evidence suggests that l i k e v i t a m i n B they affect t r a n s p o r t i n d i r e c t l y through s o m e change i n the s y n t h e s i s o r t u r n o v e r of a s t r u c t u r a l component of the cell. 6

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116 Acknowledgment

The author i s indebted to the f o l l o w i n g a s s o c i a t e s f o r a s s i s t a n c e i n c a r r y i n g out these s t u d i e s : J a n e H o l m a n , M a r i l y n M a i l e , J o s e p h u s V a n B a l g o o y , and N e d r a U t e c h . Literature

Cited


(1) Abrams, Α., in "Amino Acid Pools," by J. T. Holden, p. 764, Elsevier, Amsterdam, 1962. (2) Abrams, Α., Yamasaki, G., Redman, J., Mackenzie, C. G., Federation Proc. 19, 129 (1960). (3) Ahmed, K., Judah, J. D., Wallgren, H., Biochim. Biophys. Acta 69, 428 (1963). (4) Boezzi, J . Α., De Moss., R. D., Ibid., 49, 471 (1961). (5) Brierley, G. P., Murer, Ε., Green, D. E . , Science 140, 60 (1963). (6) Britten, R. J., McClure, F . T., Bact. Rev. 126, 292 (1962). (7) Britten, R. J., McClure, F. T., in "Amino Acid Pools," by J. T. Holden, p. 595, Elsevier, Amsterdam, 1962. (8) Christensen, H. N., in "Amino Acid Metabolism," by W. D. McElroy and B. Glass, p. 63, Johns Hopkins Press, Baltimore, 1955. (9) Christensen H. N., in "Amino Acid Pools," by J. T. Holden, p. 612, Elsevier, Amsterdam, 1962. (10) Christensen, H. N., Riggs, T. R., Coyne, Β. Α., J. Biol. Chem. 209, 413 (1954). (11) Gaby, W. L., Naughton, R. N., Logan, C., Arch. Biochem. Biophys. 82, 34 (1959). (12) Gaby, W. L., Silberman, R., Ibid., 87, 188 (1960). (13) Gaby, W. L., Wolin, H. L., Zajac, I., Cancer Res. 20, 1508 (1960). (14) Gale, E . F . , Advan. Protein. Chem. 8, 287 (1953). (15) Halvorson, H. O., Cohen, G. N., Ann. Inst. Pasteur 95, 73 (1958). (16) Hendler, R. W., in "Amino Acid Pools," by J. T. Holden, p. 750, Elsevier, Amsterdam, 1962. (17) Hendler, R. W., J. Biol. Chem. 234, 1466 (1959). (18) Hokin, L . E., Hokin, M. R., Federation Proc. 22, 8 (1963). (19) Holden, J. T., Biochim. Biophys. Acta 29, 667 (1958). (20) Ibid., 33, 581 (1959). (21) Ibid., 74, 401 (1963). (22) Holden, J . T., "Amino Acid Pools," p. 566, Elsevier, Amsterdam, 1962. (23) Holden, J. T., J. Biol. Chem. 234, 872 (1959). (24) Holden, J. T., Proc. Fifth Intern. Congr. Biochem., p. 304, Moscow, 1961. (25) Holden, J. T., unpublished manuscript. (26) Holden, J. T., Holman, J., J. Bacteriol. 73, 592 (1957). (27) Holden, J. T., Holman, J., J. Biol. Chem. 234, 865 (1959). (28) Holden, J. T., Wildman, R. B., Snell, Ε. E . , Ibid., 191, 559 (1951). (29) Hunter, G. D., Goodsall, R. Α., Biochem. J . 78, 564 (1961). (30) Hunter, G. D., James, A. T., Nature 198, 789 (1963). (31) Jacobs, F . Α., Poston, J. M . , Biochim. Biophys. Acta 51, 602 (1961). (32) Judah, J. D., Ahmed, K., McLean, Α. Ε. M . , Ibid., 65, 472 (1962). (33) Lark, K. G., Can. J. Microbiol. 5, 381 (1959). (34) Macfarlane, M. G., Nature 196, 136 (1962). (35) Mitchell, P., Ann. Rev. Microbiol. 13, 407 (1959). (36) Mitchell, P., Moyle, J., in "Bacterial Anatomy," by E. T. C. Spooner and B. A. D. Stocker, p. 150, Cambridge Univ. Press, 1956.


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(37) Mora, J., Snell, Ε. E . , Biochemistry 2, 136 (1963). (38) Oxender, D. L., Royer, M., Federation Proc. 20, 140 (1961). (39) Park, J. T., Hancock, R., J. Gen. Microbiol. 22, 249 (1960). (40) Post, R. L., Merritt, C. W., Kinsolving, C. R., Albright, C. D., J. Biol. Chem. 235, 1796 (1960). (41) Rossi, C. S., Lehninger, A. L., Biochem. Biophys. Res. Commun. 11, 411 (1963). (42) Skou, J. C., Biochim. Biophys. Acta 42, 6 (1960). (43) Weibull, C., Ann. Rev. Microbiol. 12, 1 (1958). Downloaded by UCSF LIB CKM RSCS MGMT on November 23, 2014 | http://pubs.acs.org Publication Date: January 1, 1964 | doi: 10.1021/ba-1964-0044.ch008

Received July 26, 1963. Work supported by a grant from the U. S. Public Health Service (E-1487) and a contract between the Office of Naval Research and the City of Hope Medical Center [NONR-2702(00)].


Amino Acid Transport in Bacteria - Advances in Chemistry (ACS

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