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Hexosamine formation in rat liver
VOL. 21 (1956)
PRELIMINARY NOTES
205
TABLE II ELECTROPHORETIC SEPARATION OF NUCLEOSIDE PHOSPHOTRANSFI~RASE AND P H O S P H A T A S E A solution of the purified carrot e n z y m e (Prep. I in Table I) was subjected to electrophoresis on W h a t m a n No. I p a p e r in o.i M acetate buffer of p H 5.2 (3 h, i I v / c m ) . TWO principal b a n d s were located at a b o u t + 2 cm and - - 2 cm from the origin. The eluate of the anodic fraction is designated A, t h a t of the cathodic fraction C. Conditions of a s s a y as in Table I. Incubation at 3°°
Fraction
3h
5h
~4h
O r t h o p h o s p h a t e liberated (mole °/o of donor)
A C
3° o
45 ~ I
IOO 24
Uridylic acid produced (mole % of acceptor)
A C
o 6
o II
o 26
p r e p a r a t i o n t h a t h a d been treated w i t h Celite yielded a cathodic zone producing a 13 ~o conversion to nucleotide with less t h a n 1% of o r t h o p h o s p h a t e liberated after 48 h o u r s ; on the anodic side a completely inert protein zone was observed. The e x p e r i m e n t s presented here s h o w t h a t acid p h o s p h a t a s e devoid of t r a n s f e r activity can be separated f r o m w h a t a p p e a r s to be essentially a nucleoside p h o s p h o t r a n s f e r a s e fraction of r e m a r k a b l e versatility. I n addition to producing 5'-nucleotides even from u n u s u a l nucleosides, the p h o s p h o t r a n s f e r a s e preparations, largely freed of phosphatase, as here described, a p p e a r to be able to t r a n s f e r p h o s p h a t e to the 5' position of 2'-ribonucleotides w i t h the p r o d u c t i o n of nucleoside-2",5'-diphosphates. Similarly, the cyclic ribonucleotides (nucleoside-2',3'-monohydrogen phosphates) are p h o s p h o r y l a t e d in the 5' position. F o r instance, a d e n o s i n e - 2 ' , 3 ' - m o n o h y d r o g e n p h o s p h a t e was, with p h e n y l p h o s p h a t e as the donor, converted to a compound, p r e s u m a b l y the (2':3'),5'-diphosphate, which, in four experiments, contained 1.9, 2.2, 1.9 and 2.1 g a t o m s P per mole of adenosine. These studies are being continued.
Cell Chemistry Laboratory, Department o/Biochemistry, College o/ Physicians and Surgeons, Columbia University, New York, N . Y . (U.S.A.)
MARVIN TUNIS ERWIN CHARGAFF
BRAWERMAN AND E. CHARGAFF, J . A m . Chem. Soc., 75 (1953) 2o2o, 4113 . 2 G. BRAWERMAN AND E. CHARGAFF, Biochim. Biophys. Acta, 15 (1954) 549; 16 (1955) 524.
1 G.
Received May 28th, 1956
Hexosamine formation in rat liver Glucosamine has been r e p o r t e d b y LELOIR AND CARDINI 1 to be formed in Neurospora crassa e x t r a c t s b y the following reaction: D-hexose-6-phosphate + L - g l u t a m i n e - - + n-glucosamine-6-phosphate + L-glutamate
(i)
The hexose donor was established as fructose-6-phosphate b y ROSEMAN and coworkers ~. Recently, LELOIR AND CARDINI3 showed a p a t h w a y to glucosamine f o r m a t i o n in m a m m a l i a n tissues b y a direct reaction b e t w e e n fructose-6-phosphate and NH4+ which required N-acetylglucosamine6 - p h o s p h a t e as a cofactor. I n this laboratory, an enzyme s y s t e m has been found in e x t r a c t s of r a t liver which catalyzes a reaction similar to t h a t in Neurospora ( E q u a t i o n I). NH4+ was inactive in this system. The e n z y m e was soluble after centrifuging liver e x t r a c t s m a d e in o . i 5 4 M KC1 at 18,ooo × g for 9o minutes, has a p H o p t i m u m in the range 7.4-8.0, a n d was easily inactivated, even w h e n k e p t at low t e m p e r a t u r e s . The reaction was followed b y m e a s u r e m e n t of h e x o s a m i n e f o r m a t i o n b y a slight modification of t h e m e t h o d of BLIXa after precipitation of protein w i t h 0. 4 M trichloroacetic acid. Samples were first hydrolyzed in I N HC1 at IOO° for I hour. As s h o w n in Table I,
206
PRELIMINARY NOTES
VOL. 21 (1956)
t h e r e w a s a r e q u i r e m e n t for h e x o s e - 6 - p h o s p h a t e (either g l u c o s e - 6 - p h o s p h a t e (G6P) or fructose6 - p h o s p h a t e (F6P)) a n d g l u t a m i n e . N H a + w a s ineffective as a n i t r o g e n source over a c o n c e n t r a t i o n r a n g e f r o m 5 - 2 o # M per ml. A d d i t i o n of a d e n o s i n e t r i p h o s p h a t e in t h e presence of NH4+ c a u s e d a s m a l l increase in h e x o s a m i n e f o r m a t i o n , p r e s u m a b l y v i a s y n t h e s i s of g l u t a m i n e b y reaction w i t h e n d o g e n o u s g l u t a m a t e . T h e l a t t e r alone g a v e no s y n t h e s i s . No evidence h a s b e e n f o u n d for t h e e x i s t e n c e of a n y cofactor or m e t a l r e q u i r e m e n t . E t h y l e n e d i a m i n e t e t r a a c e t a t e (5/~M/ml) h a d no effect on s y n t h e s i s f r o m G6P, b u t r e d u c e d t h a t f o r m e d f r o m g l u c o s e - l - p h o s p h a t e b y 47 °'o, p r o b a b l y b y b i n d i n g Mg++ a n d t h u s i n h i b i t i n g p h o s p h o g l u c o m u t a s e . Specific activities in t h e c r u d e s u p e r n a t a n t fluids r a n g e d b e t w e e n 0.21-o.27 i~moles/h/mg N. TABLE I HEXOSAMINE
Experimen~
I 2 3 4
FORMATION
IN
RAT
LIVER
A dditlons
EXTRACTS
Time al incubation (minutes)
Glucosamine ]ormed (t~moles)
60
0.32 0.06 0.02 0.24 o.21 0.52 0.20 o.18 0.25 0.02
G 6 P (5 IzM), g l u t a m i n e (20/~M) G 6 P (5 p M ) G l u t a m i n e (15 # M ) G 6 P (12. 5 / , M ) , g l u t a m i n e (20/zM) F 6 P (12. 5/~M), g l u t a m i n e (20 # M ) G 6 P ( i o / z M ) , g l u t a m i n e (15/~M) G 6 P ( i o / t M ) , NH4C1 (15/~M) G6P (io/,M) G 6 P (12.5/zM), g l u t a m i n e (20 # M ) G 6 P (12.5 pM), g l u t a m a t e (20/~M)
92 126 80
0.2 m l of liver s u p e r n a t a n t fluid a d d e d to each tube. I n c u b a t i o n s a t 380 in a final v o l u m e of I.o m l in p h o s p h a t e buffer (pH 7.4). A large scale p r e p a r a t i o n of t h e h e x o s a m i n e p r o d u c t w a s o b t a i n e d b y i n c u b a t i n g in a final v o l u m e of 5 ° m l in p h o s p h a t e buffer of p H 7-4, 5 o o / * m o l e s of G6P, i,ooo # m o l e s of g l u t a m i n e , a n d a 2 h o u r dialyzed fraction of r a t liver s u p e r n a t a n t fluid p r e c i p i t a t i n g b e t w e e n 1 . 7 - 2 . 3 M a m m o n i u m sulfate. A f t e r 4 h o u r s at 38°, 94/~moles of h e x o s a m i n e were formed. T h e i n c u b a t i o n was t e r m i n a t e d b y a d d i t i o n of trichloroacetic acid, a n d t h e p r o d u c t q u a n t i t a t i v e l y recovered as t h e water-soluble, alcohol-insoluble B a ++ salt, T h e p r o d u c t g a v e a color s p e c t r u m in t h e ELSONMORGAN r e a c t i o n identical w i t h t h a t of g l u c o s a m i n e a n d w a s r e s i s t a n t to acid-hydrolysis. It was a d s o r b e d v e r y w e a k l y on a io × i c m D o w e x 5 ° c a t i o n - e x c h a n g e resin (H + form), a n d e l u t e d c o m p l e t e l y w i t h distilled water. T h i s fraction d i s a p p e a r e d after h y d r o l y s i s w i t h a r a t liver micros o m a l g l u c o s e - 6 - p h o s p h a t a s e p r e p a r a t i o n , a n d t h e r e was a c o n c o m i t a n t a p p e a r a n c e of inorganic p h o s p h a t e . T h i s e n z y m e h a s b e e n r e p o r t e d to h y d r o l y z e g l u c o s a m i n e - 6 - p h o s p h a t e 5. W o r k is c o n t i n u i n g to f u r t h e r e s t a b l i s h t h e p r o d u c t s of t h i s a m i n o t r a n s f e r a s e , p r e s u m a b l y D - g l u c o s a m i n e - 6 - p h o s p h a t e a n d L - g l u t a m a t e , a n d t h e p a r t i c u l a r h e x o s e - 6 - p h o s p h a t e involved. T h i s r e a c t i o n r e p r e s e n t s a n o t h e r p a t h w a y for glucose m e t a b o l i s m in liver a n d a n o t h e r e x a m p l e of utilization of t h e a m i d e g r o u p of g l u t a m i n e as a source of a m i n o g r o u p s in s y n t h e t i c reactions.
Wilmer Institute, Johns Hopkins Medical School, Baltimore, 31d. (U.S.A.) 1 L. F. LELOIR AND C. E. CARDINI, Biochim. Biophys. Acta, 2 H. J. BLUMENTHAL, S. T. HOROWlTZ, A. HEMERLINE AND 137. 3 L. F. LELOIR AND C. E. CARDINI, Biochim. Biophys. Acta, 4 G. BLIX, Acta Chem. Scand., 2 (1948) 467 . 5 ~,. MALEY AND H. A. L A R D Y , J . Am. Chem. Soc., 78 (1956)
BURTON M. POGELL
12 (1953) 15. S. ROSEMAN, Bacteriol. Prec., (1955) 20 (1956) 331393. Received M a y I4th, 1956
PRELIMINARY NOTES
205
TABLE II ELECTROPHORETIC SEPARATION OF NUCLEOSIDE PHOSPHOTRANSFI~RASE AND P H O S P H A T A S E A solution of the purified carrot e n z y m e (Prep. I in Table I) was subjected to electrophoresis on W h a t m a n No. I p a p e r in o.i M acetate buffer of p H 5.2 (3 h, i I v / c m ) . TWO principal b a n d s were located at a b o u t + 2 cm and - - 2 cm from the origin. The eluate of the anodic fraction is designated A, t h a t of the cathodic fraction C. Conditions of a s s a y as in Table I. Incubation at 3°°
Fraction
3h
5h
~4h
O r t h o p h o s p h a t e liberated (mole °/o of donor)
A C
3° o
45 ~ I
IOO 24
Uridylic acid produced (mole % of acceptor)
A C
o 6
o II
o 26
p r e p a r a t i o n t h a t h a d been treated w i t h Celite yielded a cathodic zone producing a 13 ~o conversion to nucleotide with less t h a n 1% of o r t h o p h o s p h a t e liberated after 48 h o u r s ; on the anodic side a completely inert protein zone was observed. The e x p e r i m e n t s presented here s h o w t h a t acid p h o s p h a t a s e devoid of t r a n s f e r activity can be separated f r o m w h a t a p p e a r s to be essentially a nucleoside p h o s p h o t r a n s f e r a s e fraction of r e m a r k a b l e versatility. I n addition to producing 5'-nucleotides even from u n u s u a l nucleosides, the p h o s p h o t r a n s f e r a s e preparations, largely freed of phosphatase, as here described, a p p e a r to be able to t r a n s f e r p h o s p h a t e to the 5' position of 2'-ribonucleotides w i t h the p r o d u c t i o n of nucleoside-2",5'-diphosphates. Similarly, the cyclic ribonucleotides (nucleoside-2',3'-monohydrogen phosphates) are p h o s p h o r y l a t e d in the 5' position. F o r instance, a d e n o s i n e - 2 ' , 3 ' - m o n o h y d r o g e n p h o s p h a t e was, with p h e n y l p h o s p h a t e as the donor, converted to a compound, p r e s u m a b l y the (2':3'),5'-diphosphate, which, in four experiments, contained 1.9, 2.2, 1.9 and 2.1 g a t o m s P per mole of adenosine. These studies are being continued.
Cell Chemistry Laboratory, Department o/Biochemistry, College o/ Physicians and Surgeons, Columbia University, New York, N . Y . (U.S.A.)
MARVIN TUNIS ERWIN CHARGAFF
BRAWERMAN AND E. CHARGAFF, J . A m . Chem. Soc., 75 (1953) 2o2o, 4113 . 2 G. BRAWERMAN AND E. CHARGAFF, Biochim. Biophys. Acta, 15 (1954) 549; 16 (1955) 524.
1 G.
Received May 28th, 1956
Hexosamine formation in rat liver Glucosamine has been r e p o r t e d b y LELOIR AND CARDINI 1 to be formed in Neurospora crassa e x t r a c t s b y the following reaction: D-hexose-6-phosphate + L - g l u t a m i n e - - + n-glucosamine-6-phosphate + L-glutamate
(i)
The hexose donor was established as fructose-6-phosphate b y ROSEMAN and coworkers ~. Recently, LELOIR AND CARDINI3 showed a p a t h w a y to glucosamine f o r m a t i o n in m a m m a l i a n tissues b y a direct reaction b e t w e e n fructose-6-phosphate and NH4+ which required N-acetylglucosamine6 - p h o s p h a t e as a cofactor. I n this laboratory, an enzyme s y s t e m has been found in e x t r a c t s of r a t liver which catalyzes a reaction similar to t h a t in Neurospora ( E q u a t i o n I). NH4+ was inactive in this system. The e n z y m e was soluble after centrifuging liver e x t r a c t s m a d e in o . i 5 4 M KC1 at 18,ooo × g for 9o minutes, has a p H o p t i m u m in the range 7.4-8.0, a n d was easily inactivated, even w h e n k e p t at low t e m p e r a t u r e s . The reaction was followed b y m e a s u r e m e n t of h e x o s a m i n e f o r m a t i o n b y a slight modification of t h e m e t h o d of BLIXa after precipitation of protein w i t h 0. 4 M trichloroacetic acid. Samples were first hydrolyzed in I N HC1 at IOO° for I hour. As s h o w n in Table I,
206
PRELIMINARY NOTES
VOL. 21 (1956)
t h e r e w a s a r e q u i r e m e n t for h e x o s e - 6 - p h o s p h a t e (either g l u c o s e - 6 - p h o s p h a t e (G6P) or fructose6 - p h o s p h a t e (F6P)) a n d g l u t a m i n e . N H a + w a s ineffective as a n i t r o g e n source over a c o n c e n t r a t i o n r a n g e f r o m 5 - 2 o # M per ml. A d d i t i o n of a d e n o s i n e t r i p h o s p h a t e in t h e presence of NH4+ c a u s e d a s m a l l increase in h e x o s a m i n e f o r m a t i o n , p r e s u m a b l y v i a s y n t h e s i s of g l u t a m i n e b y reaction w i t h e n d o g e n o u s g l u t a m a t e . T h e l a t t e r alone g a v e no s y n t h e s i s . No evidence h a s b e e n f o u n d for t h e e x i s t e n c e of a n y cofactor or m e t a l r e q u i r e m e n t . E t h y l e n e d i a m i n e t e t r a a c e t a t e (5/~M/ml) h a d no effect on s y n t h e s i s f r o m G6P, b u t r e d u c e d t h a t f o r m e d f r o m g l u c o s e - l - p h o s p h a t e b y 47 °'o, p r o b a b l y b y b i n d i n g Mg++ a n d t h u s i n h i b i t i n g p h o s p h o g l u c o m u t a s e . Specific activities in t h e c r u d e s u p e r n a t a n t fluids r a n g e d b e t w e e n 0.21-o.27 i~moles/h/mg N. TABLE I HEXOSAMINE
Experimen~
I 2 3 4
FORMATION
IN
RAT
LIVER
A dditlons
EXTRACTS
Time al incubation (minutes)
Glucosamine ]ormed (t~moles)
60
0.32 0.06 0.02 0.24 o.21 0.52 0.20 o.18 0.25 0.02
G 6 P (5 IzM), g l u t a m i n e (20/~M) G 6 P (5 p M ) G l u t a m i n e (15 # M ) G 6 P (12. 5 / , M ) , g l u t a m i n e (20/zM) F 6 P (12. 5/~M), g l u t a m i n e (20 # M ) G 6 P ( i o / z M ) , g l u t a m i n e (15/~M) G 6 P ( i o / t M ) , NH4C1 (15/~M) G6P (io/,M) G 6 P (12.5/zM), g l u t a m i n e (20 # M ) G 6 P (12.5 pM), g l u t a m a t e (20/~M)
92 126 80
0.2 m l of liver s u p e r n a t a n t fluid a d d e d to each tube. I n c u b a t i o n s a t 380 in a final v o l u m e of I.o m l in p h o s p h a t e buffer (pH 7.4). A large scale p r e p a r a t i o n of t h e h e x o s a m i n e p r o d u c t w a s o b t a i n e d b y i n c u b a t i n g in a final v o l u m e of 5 ° m l in p h o s p h a t e buffer of p H 7-4, 5 o o / * m o l e s of G6P, i,ooo # m o l e s of g l u t a m i n e , a n d a 2 h o u r dialyzed fraction of r a t liver s u p e r n a t a n t fluid p r e c i p i t a t i n g b e t w e e n 1 . 7 - 2 . 3 M a m m o n i u m sulfate. A f t e r 4 h o u r s at 38°, 94/~moles of h e x o s a m i n e were formed. T h e i n c u b a t i o n was t e r m i n a t e d b y a d d i t i o n of trichloroacetic acid, a n d t h e p r o d u c t q u a n t i t a t i v e l y recovered as t h e water-soluble, alcohol-insoluble B a ++ salt, T h e p r o d u c t g a v e a color s p e c t r u m in t h e ELSONMORGAN r e a c t i o n identical w i t h t h a t of g l u c o s a m i n e a n d w a s r e s i s t a n t to acid-hydrolysis. It was a d s o r b e d v e r y w e a k l y on a io × i c m D o w e x 5 ° c a t i o n - e x c h a n g e resin (H + form), a n d e l u t e d c o m p l e t e l y w i t h distilled water. T h i s fraction d i s a p p e a r e d after h y d r o l y s i s w i t h a r a t liver micros o m a l g l u c o s e - 6 - p h o s p h a t a s e p r e p a r a t i o n , a n d t h e r e was a c o n c o m i t a n t a p p e a r a n c e of inorganic p h o s p h a t e . T h i s e n z y m e h a s b e e n r e p o r t e d to h y d r o l y z e g l u c o s a m i n e - 6 - p h o s p h a t e 5. W o r k is c o n t i n u i n g to f u r t h e r e s t a b l i s h t h e p r o d u c t s of t h i s a m i n o t r a n s f e r a s e , p r e s u m a b l y D - g l u c o s a m i n e - 6 - p h o s p h a t e a n d L - g l u t a m a t e , a n d t h e p a r t i c u l a r h e x o s e - 6 - p h o s p h a t e involved. T h i s r e a c t i o n r e p r e s e n t s a n o t h e r p a t h w a y for glucose m e t a b o l i s m in liver a n d a n o t h e r e x a m p l e of utilization of t h e a m i d e g r o u p of g l u t a m i n e as a source of a m i n o g r o u p s in s y n t h e t i c reactions.
Wilmer Institute, Johns Hopkins Medical School, Baltimore, 31d. (U.S.A.) 1 L. F. LELOIR AND C. E. CARDINI, Biochim. Biophys. Acta, 2 H. J. BLUMENTHAL, S. T. HOROWlTZ, A. HEMERLINE AND 137. 3 L. F. LELOIR AND C. E. CARDINI, Biochim. Biophys. Acta, 4 G. BLIX, Acta Chem. Scand., 2 (1948) 467 . 5 ~,. MALEY AND H. A. L A R D Y , J . Am. Chem. Soc., 78 (1956)
BURTON M. POGELL
12 (1953) 15. S. ROSEMAN, Bacteriol. Prec., (1955) 20 (1956) 331393. Received M a y I4th, 1956