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The metabolism of l -rhamnose by Escherichia coli
voL. 17 (z955)
SHORT COMMUNICATIONS, PRELIMINARY NOTES
289
The metabolism of L-rhamnose by Escherichia coli In this note are described p r e l i m i n a r y studies on two euzymic steps in the metabolism of L-rhamnose by Escherichia coll. Washed ceil suspensions of E. coli, strain B, w h e n g r o w n in the presence of rhamnose, fermented this sugar with the p r o d u c t i o n of acid and small a m o u n t s of gas. W h e n w a t e r e x t r a c t s (dialyzed 3-4 hours) of acetone p o w d e r s of these cells were incubated anaerobically with r h a m n o s e and the reaction m i x t u r e analyzed, a ketose was detected b y m e a n s of the cysteine-carbazole test 1. Paper c h r o m a t o g r a p h i c techniques showed t h a t this material had a greater R F value t h a n r h a m n o s e in the solvent m i x t u r e s b u t a n o l - p y r i d i n e - w a t e r (3:2:1-51 and b u t a n o l - e t h a n o l - w a t e r (5: I:4). W h e n eluted from the papers it gave positive t e s t s for ketose (abs. max. 545 mlQ, m e t h y l pentose 2 (abs. max. 4oo mp) and reducing sugar a. The orcinol test for pentoses was negative. F r o m the results of these tests it would a p p e a r likely t h a t the c o m p o u n d formed is r h a m n o ketose. This h y p o t h e s i s is f u r t h e r s u p p o r t e d b y the fact t h a t the ketose p r e p a r e d by refluxing Lr h a m n o s e with pyridine, a c o m m o n m e t h o d for the p r e p a r a t i o n of ketoses from their respective aldoses, has similar properties to the ketose obtained in these experiments. The addition of adenosine t r i p h o s p h a t e (ATP) and r h a m n o s e to such dialyzed acetone p o w d e r e x t r a c t s of E. coli resulted in the f o r m a t i o n of a p h o s p h o r y l a t e d c o m p o u n d as determined by the m a n o m e t r i c naethod of COLOWlCK AND K A L C K A R 4. I n Table I are s h o w n the results obtained with the cysteine-carbazole test on the s u p e r n a t a n t s of samples of the reaction m i x t u r e s treated with either Ba(OH)2 and ZnSOa, or w i t h trichloracetic acid. The ketose which formed in the vessel containing rhamnose, ATP and e x t r a c t was a h n o s t completely removed b y the B a ( O H ) 2 - ZnSO 4 t r e a t m e n t which precipitates p h o s p h o r y l a t e d c o m p o u n d s as well as. proteins. The results of this e x p e r i m e n t suggested t h a t in the presence of ATP, rhanlnoketose p h o s p h a t e w a s p r o b a b l y formed. TAI3I,E I K E T O S E P H O S P H A T E FORMATION FROM RHAMNOSE BY E s c h e r i c h i a
coli
Colorimeter readings at 545 m # in the cysteine-carbazole test per o . i m l o / s a m p l e Incubation system
Initial Ba(OH) 2 + ZnSO 4
1. 2. 34.
R h a m n o s e + ATP + e x t r a c t Rhamnose + extract ATP + extract R h a m n o s e + ATP
0.030 o.o21 o.o16 o.o 3I
Final TCA**
Ba(OH) 2 + ZnSO ~
T C A **
0.043
0.059
0.604
O.OI8
O.511
0.530
o.o25 o.o39
O.Ol 4 o.o28
0.024 o.o34
The complete s y s t e m contained 0.002 3 I L-rhamnose, 0.0033 111 ATP, 0.025 M N a H C O a and i ml of extract. Total v o l u m e 3 mh Time of i n c u b a t i o n 1. 5 hours. T e m p e r a t u r e 33 °. \ V a r b u r g flasks were gassed with a m i x t u r e of 5 % COz and 95 % N2 before t i p p i n g the ATP and rhamnose. * Duplicate cups to which protein p r e c i p i t a n t s were added at the s t a r t of the e x p e r i m e n t represent the initial values shown. ** Trichloracetic acid.
W h e n the newly formed p h o s p h a t e c o m p o u n d was separated by m e a n s of p a p e r c h r o m a t o g r a p h y and eluted from the p a p e r it gave positive tests for b o u n d p h o s p h o r u s , ketose, m e t h y l pentose and reducing sugar. The material was 8 o - 9 o % hydrolyzed after io m i n u t e s at IOO° in N HC1, which suggests t h a t this c o m p o u n d m a y be r h a m n o k e t o s e - i - p h o s p h a t e . D. M. WILSON Department o[ Bacteriology, Communicable Diseases Division, S. AJL
A r m y Medical Service Graduate School, Washington, D.C. (U.S.A.) 1 Z. DlSCHE AND E. BORENFREUND, J. Biol. Chem., I92 (1951) 583 . 2 Z. DISCHE AND L. B. SHETTLES, J. Biol. Chem., 175 (1948) 595a M. SOMOGYI, J. Biol. Chem., I6O (I945) 61. 4 S. P. COLOWlCK .aND H. M. KALCKAR, J. Biol. Chem., 148 (I943) 117. Received April ~5th, 1955
SHORT COMMUNICATIONS, PRELIMINARY NOTES
289
The metabolism of L-rhamnose by Escherichia coli In this note are described p r e l i m i n a r y studies on two euzymic steps in the metabolism of L-rhamnose by Escherichia coll. Washed ceil suspensions of E. coli, strain B, w h e n g r o w n in the presence of rhamnose, fermented this sugar with the p r o d u c t i o n of acid and small a m o u n t s of gas. W h e n w a t e r e x t r a c t s (dialyzed 3-4 hours) of acetone p o w d e r s of these cells were incubated anaerobically with r h a m n o s e and the reaction m i x t u r e analyzed, a ketose was detected b y m e a n s of the cysteine-carbazole test 1. Paper c h r o m a t o g r a p h i c techniques showed t h a t this material had a greater R F value t h a n r h a m n o s e in the solvent m i x t u r e s b u t a n o l - p y r i d i n e - w a t e r (3:2:1-51 and b u t a n o l - e t h a n o l - w a t e r (5: I:4). W h e n eluted from the papers it gave positive t e s t s for ketose (abs. max. 545 mlQ, m e t h y l pentose 2 (abs. max. 4oo mp) and reducing sugar a. The orcinol test for pentoses was negative. F r o m the results of these tests it would a p p e a r likely t h a t the c o m p o u n d formed is r h a m n o ketose. This h y p o t h e s i s is f u r t h e r s u p p o r t e d b y the fact t h a t the ketose p r e p a r e d by refluxing Lr h a m n o s e with pyridine, a c o m m o n m e t h o d for the p r e p a r a t i o n of ketoses from their respective aldoses, has similar properties to the ketose obtained in these experiments. The addition of adenosine t r i p h o s p h a t e (ATP) and r h a m n o s e to such dialyzed acetone p o w d e r e x t r a c t s of E. coli resulted in the f o r m a t i o n of a p h o s p h o r y l a t e d c o m p o u n d as determined by the m a n o m e t r i c naethod of COLOWlCK AND K A L C K A R 4. I n Table I are s h o w n the results obtained with the cysteine-carbazole test on the s u p e r n a t a n t s of samples of the reaction m i x t u r e s treated with either Ba(OH)2 and ZnSOa, or w i t h trichloracetic acid. The ketose which formed in the vessel containing rhamnose, ATP and e x t r a c t was a h n o s t completely removed b y the B a ( O H ) 2 - ZnSO 4 t r e a t m e n t which precipitates p h o s p h o r y l a t e d c o m p o u n d s as well as. proteins. The results of this e x p e r i m e n t suggested t h a t in the presence of ATP, rhanlnoketose p h o s p h a t e w a s p r o b a b l y formed. TAI3I,E I K E T O S E P H O S P H A T E FORMATION FROM RHAMNOSE BY E s c h e r i c h i a
coli
Colorimeter readings at 545 m # in the cysteine-carbazole test per o . i m l o / s a m p l e Incubation system
Initial Ba(OH) 2 + ZnSO 4
1. 2. 34.
R h a m n o s e + ATP + e x t r a c t Rhamnose + extract ATP + extract R h a m n o s e + ATP
0.030 o.o21 o.o16 o.o 3I
Final TCA**
Ba(OH) 2 + ZnSO ~
T C A **
0.043
0.059
0.604
O.OI8
O.511
0.530
o.o25 o.o39
O.Ol 4 o.o28
0.024 o.o34
The complete s y s t e m contained 0.002 3 I L-rhamnose, 0.0033 111 ATP, 0.025 M N a H C O a and i ml of extract. Total v o l u m e 3 mh Time of i n c u b a t i o n 1. 5 hours. T e m p e r a t u r e 33 °. \ V a r b u r g flasks were gassed with a m i x t u r e of 5 % COz and 95 % N2 before t i p p i n g the ATP and rhamnose. * Duplicate cups to which protein p r e c i p i t a n t s were added at the s t a r t of the e x p e r i m e n t represent the initial values shown. ** Trichloracetic acid.
W h e n the newly formed p h o s p h a t e c o m p o u n d was separated by m e a n s of p a p e r c h r o m a t o g r a p h y and eluted from the p a p e r it gave positive tests for b o u n d p h o s p h o r u s , ketose, m e t h y l pentose and reducing sugar. The material was 8 o - 9 o % hydrolyzed after io m i n u t e s at IOO° in N HC1, which suggests t h a t this c o m p o u n d m a y be r h a m n o k e t o s e - i - p h o s p h a t e . D. M. WILSON Department o[ Bacteriology, Communicable Diseases Division, S. AJL
A r m y Medical Service Graduate School, Washington, D.C. (U.S.A.) 1 Z. DlSCHE AND E. BORENFREUND, J. Biol. Chem., I92 (1951) 583 . 2 Z. DISCHE AND L. B. SHETTLES, J. Biol. Chem., 175 (1948) 595a M. SOMOGYI, J. Biol. Chem., I6O (I945) 61. 4 S. P. COLOWlCK .aND H. M. KALCKAR, J. Biol. Chem., 148 (I943) 117. Received April ~5th, 1955