- Email: [email protected]
β-Alanine-α-alanine transaminase of Pseudomonas
VOL. 33 (1959)
PRELIMINARY I~OTES
591
From the specific activities of different RNA fractions the relative amounts of pRNA and rRNA could be calculated, and they proved to agree very well with the values obtained by direct determination. The composition of RNA was characteristic of the tissues: the content of rRNA was about 30 % in lymphatic cells and about 15 % in the liver; the rest was pRNA. The nucleotide compositions of pRNA and rRNA differ quite markedly, the content of guanylic acid being higher in the former than in the latter. It appears that the phenol method of KIRBY1 roughly fractionates two classes of RNA differing in function in the cell. Like DNA, the bulk of pRNA which is insoluble in I M NaC1 may be duplicated only during the cell division cycle (c/. HOTTA AND OSAWAa), and is metabolically rather inert. On the other hand, rRNA is metabolically very active, turning over at a very high rate. The mode of linkage of RNA to protein must be quite different for the two fractions of RNA, since only pRNA can be released by aqueous phenol. Distribution of the two forms of RNA among different subcellular fractions is now being explored. This work was supported in part by grants from the Scientific Research Funds of the Ministry of Education. We are grateful to Dr. Y. SUGINO, Takeda Pharmaceutical Industries, Dr. S. OSAWA,Nagoya University, and Dr. S. IWANAGA, Kyoto University, for their advice and help. A . SIBATANI
Cytochemistry Laboratory, Yamaguti Medical School, Ube (Japan)
K. YAMANA* K. KIMURA H. OKAGAKI
1 K. S. KIRBY, Biochem. J., 64 (1956) 405. R. A. BROWN, M. C. DAVIES, J. S. COLTER, J. B. LOGAN AND D. KRITCHEVSKY, Proc. Natl. Acad. Sci. U.S., 43 (1957) 857. a y . HOTTA AND S. OSAWA,Biochim. Biophys. Acta, 28 (1958) 642. 4 A. SIBATANI,Exptl. Cell Research, in t h e press. 5 A. SIBATANI, Biochim. Biophys. Acta, 25 (1957) 592. 6 S. OSAWA, K. TAKATA AND Y. HOTTA, Biochim. Biophys. Acta, 28 (1958) 271. 7 E. S. CANELLAKIS AND R. MANTSAVINOS, Biochim. Biophys. Aeta, 27 (1958) 643.
Received March 3rd, 1959 * O n leave f r o m t h e D e p a r t m e n t of Biology, F a c u l t y of Science, K y u s h u U n i v e r s i t y , F u k u o k a .
fl-Alanine-a-alanine transaminase of Pseudornonas Several papers have appeared in the literature which indicated transamination of /3-alaninel-L but the exact nature of the reaction as well as the products have not been completely elucidated. A partially purified enzyme preparation from Pseudomonas sp. (No. 23) is now shown to catalyze the transamination of /3-alanine with pyruvic acid, resulting in the formation of stoichiometric quantities of malonic semiaidehyde and a-alanine. Cells were grown and extracts were made as described previously s except that fl-alanine was used as a major carbon and nitrogen source in the growth media. The enzyme activity was assayed by the appearance of malonic semialdehyde using a colorimetric reaction with diazotized p-nitroaniline 4. The enzyme was purified about 20 fold by heat treatment (IO min at 6o°), followed by fractionation with (NH4)2SO4 and dialysis against 0.02 M potassium phosphate, pH 8.0, for 3 h at o °.
TABLE I
+
--
0.3
-
--
--
-
--
0.2
0.3
16.o
A Pyruvic acid
All t h e n u m b e r s are e x p r e s s e d in #,moles. * H e a t e d in b o i l i n g w a t e r for IO min. ** C a l c u l a t e d f r om o x a l o a c e t a t e as s t a n d a r d .
Complete minus p y r u v a t e
Complete minus/~-alanine
--
C o m p l e t e w i t h boiled e n z y m e *
0.8
- - 15.o
it fl-Alanine
Complete
System
o.o
o.o
o.o
+ 16. 5
Ninkydrin assay
o.o
o.o
o.o
+ 16.3
Bioacsay
z] a-Alanine
+
+
+
0.2
0.2
0.5
+ 17.o
p-nitroaniline assayS*
+
--
o.I
0. 4
o.o
+ 15.5
assay
As CO, A niline citrate
+
o.o
o.o
0.2
+ 13.8
p-Hydroxydiphenyl assay
As acetaldehyde
o.o
o.o
o.o
+ 16.1
As 2,4-dinitrophenylkydracone
A Malonic scmialdekyde
o.o
o.o
o.o
+ 16.6
Enzymic assay
T h e c o m p l e t e i n c u b a t i o n m i x t u r e c o n t a i n e d 90 /*moles fl-alanine, 8o /*moles s o d i u m p y r u v a t e , 600 /*moles p o t a s s i u m p h o s p h a t e , p H 8.0, a n d 1.2 m g e n z y m e in a final vol. of 6.0 ml. A f t e r t h e i n c u b a t i o n w a s c a r r i e d o u t for 4 ° rain a t 35 °, t h e r e a c t i o n w a s s t o p p e d w i t h 0.6 m l IO % HC104. P y r u v i c aci d w as d e t e r m i n e d s p e c t r o p h o t o m e t r i c a l l y w i t h r e d u c e d d i p h o s p h o p y r i d i n e n u c l e o t i d e a n d c r y s t a l l i n e l a c t i c d e h y d r o g e n a s e 5. Malonic s e m i a l d e h y d e was d e t e r m i n e d m a n o m e t r i c a l l y w i t h a n i l i n e c i t r a t e e, a n d c o l o r i m e t r i c a l l y w i t h d i a z o t i z e d p - n i t r o a n i l i n e . M alo n ic s e m i a l d e h y d e w as c o n v e r t e d to a c e t a l d e h y d e b y s t e a m d i s t i l l a t i o n of t h e acidified r e a c t i o n m i x t u r e a n d w a s d e t e r m i n e d c o l o r i m e t r i c a l l y w i t h p - h y d r o x y d i p h e n y l ~ or as t h e 2 , 4 - d i n i t r o p h e n y l h y d r a z o n e , a n d also s p e c t r o p h o t o m e t r i c a l l y w i t h r e d u c e d d i p h o s p h o p y r i d i n e n u c l e o t i d e a n d c r y s t a l l i n e y e a s t alco h o l deh y d r o g e n a s e 8. a - A l a n i n e a n d f l- ala nin e we re d e t e r m i n e d c o l o r i m e t r i c a l l y w i t h n i n h y d r i n a f t e r s e p a r a t i o n b y i o n - e x c h a n g e c h r o m a t o g r a p h y w i t h a Dowex-5o c o l u m n ~. a - A l a n i n e w a s also d e t e r m i n e d m i c r o b i o l o g i c a l l y u s i n g L e u c o n o s t o c c i t r o v o r u m 8o8110 a n d t h e H e n d e r s o n - S n e l l m e d i u m 11 w h i c h c o n t a i n e d l e u c o v o r i n . (We a re i n d e b t e d t o Miss S. ISHIDA a n d Dr. T. SUZUKI for t h i s assay.)
THE STOICHIOMETRY OF THE R~ACTION
ko
0
0 B'I
to
voL. 33 (1959)
PRELIMINARY NOTES
593
The stoichiometry of the reaction is shown inTable I. a-Alanine was also identified by paper chromatography using five different solvent systems*. Malonic semialdehyde was isolated from a large-scale incubation mixture by ether extraction after acidification of the solution, and was identified as 2,4-dinitrophenylhydrazone by paper chromatography using three different solvent systems by comparison with an authentic sample**. Further evidence for the identity was provided by the quantitative decarboxylation with aniline citrate and by the production of equivalent amount of acetaldehyde upon steam distillation from the acidified reaction mixture. Acetaldehyde was also identified as the 2,4-dinitrophenylhydrazone. The reaction appears to be freely reversible, since the enzyme preparation catalyzes the stoichiometric formation of pyruvic acid and fl-alanine from a-alanine and malonic semialdehyde. Pyruvic acid acts as specific amino acceptor in the forward reaction. Neither a-ketoglutarate, fl-ketoglutarate, ~-ketoadipate nor oxaloacetate could replace pyruvate as amino acceptor. However, 7-aminobutyrate acts as amino donor to some extent. Preliminary evidence indicates that pyridoxal phosphate stimulates the activity of the dialyzed enzyme preparation. Studies are now in progress in order to elucidate the further metabolism of malonic semialdehyde. This investigation was supported in part by research grants from the National Institutes of Health (C-4222), the Rockefeller Foundation and the Jane Coffin Childs Memorial Fund for Medical Research. YASUTOMI N I S H I Z U K A
Depart~nent of Medical Cher~istry, Kyoto University Fa~ultv o/Medicine, Kyoto (Japan)
MASAZUMI T A K E S H I T A SIGERU
KUNO
OSAMU H A Y A I S H I 1 E. ROBERTS AND H. M. BREGOFF, J. Biol. Chem., 2Ol (1953) 393. 2 E. ROBERTS, P. AYENGER AND I. POSNER, J. Biol. Chem., '203 (1953) 195. 3 0 . HAYAISHI, M. KATAGIRI AND S. ROTHBERG, J. Biol. Chem., 229 (1957) 905. 4 p. G. WALKER, Biochem. J., 58 (1954) 699. s A. KORNBERG, J. Biol. Chem., 182 (195 o) 805. s N. L. EDSON, Biochem. J., 29 (1953) 2o82. E. EEGEIWE, Z. anal. Chem., 95 (1933) 323 • s E. RACKEE, J. Biol. Chem., 184 (195 o) 313 . 9 S. MOORE AND W. H. STEIN, ]. Biol. Chem., 176 (1948) 367 . 10 H . E. SAUBERLICH, J. Biol. Chem., 177 (1949) 177. 11 L. M. HENDERSON AND E. E. SNELL, J. Biol. Chem., 172 (1948) 15. 12 A. WOHL AND W . EMMERICH, Bet., 33 (19oo) 276o.
Received March 3Ist, 1959 * P a p e r c h r o m a t o g r a p h y w a s carried o u t on W h a t m a n No. i paper. Rp v a l u e s for a - a l a n i n e a n d t5-alanine w i t h i s o p r o p a n o l - w a t e r (8:2) were 0.33 a n d 0.25; w i t h e t h a n o l - a m m o n i a - w a t e r (18 :I :I) o.23 a n d o.I 1 ; w i t h n - b u t a n o l - p y r i d i n e - w a t e r (1:1:1) 0.43 a n d o.34; w i t h p h e n o l - w a t e r (8 :,2,) o.52 a n d 0.62; w i t h n - b u t a n o l - a c e t i c a c i d - w a t e r (4:2:3) 0.27 a n d o.33, respectively. T h e a u t h e n t i c s a m p l e w a s p r e p a r e d f r o m malonic s e m i a l d e h y d e w h i c h w a s s y n t h e s i z e d c h e m i c a l l y TM. W e are i n d e b t e d to Mr. H. KONDO, Drs. K. HAYASHI a n d T. SUZUKI for t h e chemical s y n t h e s i s of m a l o n i c s e m i a l d e h y d e . P a p e r c h r o m a t o g r a p h y w a s carried o u t on W h a t m a n No. I p a p e r a n d s p r a y e d w i t h dilute alkali. RF v a l u e s for t h e 2 , 4 - d i n i t r o p h e n y l h y d r a z o n e s of malonic s e m i a l d e h y d e a n d p y r u v i c acid w i t h i s o p r o p a n o l - i s o a m y l a l c o h o l - p y r i d i n e - w a t e r (2o: 4 :1:5) were 0.56 a n d 0.45; w i t h i s o a m y l a l c o h o l - e t h a n o l - o . i M c a r b o n a t e , p H lO. 7 (5:I:2, u p p e r layer) o.31 a n d o . i 3 ; w i t h n - b u t a n o l - e t h a n o l - o . I M c a r b o n a t e , p H lO. 7 (6:1 :i, lower layer) o.65 a n d o.56, respectively.
PRELIMINARY I~OTES
591
From the specific activities of different RNA fractions the relative amounts of pRNA and rRNA could be calculated, and they proved to agree very well with the values obtained by direct determination. The composition of RNA was characteristic of the tissues: the content of rRNA was about 30 % in lymphatic cells and about 15 % in the liver; the rest was pRNA. The nucleotide compositions of pRNA and rRNA differ quite markedly, the content of guanylic acid being higher in the former than in the latter. It appears that the phenol method of KIRBY1 roughly fractionates two classes of RNA differing in function in the cell. Like DNA, the bulk of pRNA which is insoluble in I M NaC1 may be duplicated only during the cell division cycle (c/. HOTTA AND OSAWAa), and is metabolically rather inert. On the other hand, rRNA is metabolically very active, turning over at a very high rate. The mode of linkage of RNA to protein must be quite different for the two fractions of RNA, since only pRNA can be released by aqueous phenol. Distribution of the two forms of RNA among different subcellular fractions is now being explored. This work was supported in part by grants from the Scientific Research Funds of the Ministry of Education. We are grateful to Dr. Y. SUGINO, Takeda Pharmaceutical Industries, Dr. S. OSAWA,Nagoya University, and Dr. S. IWANAGA, Kyoto University, for their advice and help. A . SIBATANI
Cytochemistry Laboratory, Yamaguti Medical School, Ube (Japan)
K. YAMANA* K. KIMURA H. OKAGAKI
1 K. S. KIRBY, Biochem. J., 64 (1956) 405. R. A. BROWN, M. C. DAVIES, J. S. COLTER, J. B. LOGAN AND D. KRITCHEVSKY, Proc. Natl. Acad. Sci. U.S., 43 (1957) 857. a y . HOTTA AND S. OSAWA,Biochim. Biophys. Acta, 28 (1958) 642. 4 A. SIBATANI,Exptl. Cell Research, in t h e press. 5 A. SIBATANI, Biochim. Biophys. Acta, 25 (1957) 592. 6 S. OSAWA, K. TAKATA AND Y. HOTTA, Biochim. Biophys. Acta, 28 (1958) 271. 7 E. S. CANELLAKIS AND R. MANTSAVINOS, Biochim. Biophys. Aeta, 27 (1958) 643.
Received March 3rd, 1959 * O n leave f r o m t h e D e p a r t m e n t of Biology, F a c u l t y of Science, K y u s h u U n i v e r s i t y , F u k u o k a .
fl-Alanine-a-alanine transaminase of Pseudornonas Several papers have appeared in the literature which indicated transamination of /3-alaninel-L but the exact nature of the reaction as well as the products have not been completely elucidated. A partially purified enzyme preparation from Pseudomonas sp. (No. 23) is now shown to catalyze the transamination of /3-alanine with pyruvic acid, resulting in the formation of stoichiometric quantities of malonic semiaidehyde and a-alanine. Cells were grown and extracts were made as described previously s except that fl-alanine was used as a major carbon and nitrogen source in the growth media. The enzyme activity was assayed by the appearance of malonic semialdehyde using a colorimetric reaction with diazotized p-nitroaniline 4. The enzyme was purified about 20 fold by heat treatment (IO min at 6o°), followed by fractionation with (NH4)2SO4 and dialysis against 0.02 M potassium phosphate, pH 8.0, for 3 h at o °.
TABLE I
+
--
0.3
-
--
--
-
--
0.2
0.3
16.o
A Pyruvic acid
All t h e n u m b e r s are e x p r e s s e d in #,moles. * H e a t e d in b o i l i n g w a t e r for IO min. ** C a l c u l a t e d f r om o x a l o a c e t a t e as s t a n d a r d .
Complete minus p y r u v a t e
Complete minus/~-alanine
--
C o m p l e t e w i t h boiled e n z y m e *
0.8
- - 15.o
it fl-Alanine
Complete
System
o.o
o.o
o.o
+ 16. 5
Ninkydrin assay
o.o
o.o
o.o
+ 16.3
Bioacsay
z] a-Alanine
+
+
+
0.2
0.2
0.5
+ 17.o
p-nitroaniline assayS*
+
--
o.I
0. 4
o.o
+ 15.5
assay
As CO, A niline citrate
+
o.o
o.o
0.2
+ 13.8
p-Hydroxydiphenyl assay
As acetaldehyde
o.o
o.o
o.o
+ 16.1
As 2,4-dinitrophenylkydracone
A Malonic scmialdekyde
o.o
o.o
o.o
+ 16.6
Enzymic assay
T h e c o m p l e t e i n c u b a t i o n m i x t u r e c o n t a i n e d 90 /*moles fl-alanine, 8o /*moles s o d i u m p y r u v a t e , 600 /*moles p o t a s s i u m p h o s p h a t e , p H 8.0, a n d 1.2 m g e n z y m e in a final vol. of 6.0 ml. A f t e r t h e i n c u b a t i o n w a s c a r r i e d o u t for 4 ° rain a t 35 °, t h e r e a c t i o n w a s s t o p p e d w i t h 0.6 m l IO % HC104. P y r u v i c aci d w as d e t e r m i n e d s p e c t r o p h o t o m e t r i c a l l y w i t h r e d u c e d d i p h o s p h o p y r i d i n e n u c l e o t i d e a n d c r y s t a l l i n e l a c t i c d e h y d r o g e n a s e 5. Malonic s e m i a l d e h y d e was d e t e r m i n e d m a n o m e t r i c a l l y w i t h a n i l i n e c i t r a t e e, a n d c o l o r i m e t r i c a l l y w i t h d i a z o t i z e d p - n i t r o a n i l i n e . M alo n ic s e m i a l d e h y d e w as c o n v e r t e d to a c e t a l d e h y d e b y s t e a m d i s t i l l a t i o n of t h e acidified r e a c t i o n m i x t u r e a n d w a s d e t e r m i n e d c o l o r i m e t r i c a l l y w i t h p - h y d r o x y d i p h e n y l ~ or as t h e 2 , 4 - d i n i t r o p h e n y l h y d r a z o n e , a n d also s p e c t r o p h o t o m e t r i c a l l y w i t h r e d u c e d d i p h o s p h o p y r i d i n e n u c l e o t i d e a n d c r y s t a l l i n e y e a s t alco h o l deh y d r o g e n a s e 8. a - A l a n i n e a n d f l- ala nin e we re d e t e r m i n e d c o l o r i m e t r i c a l l y w i t h n i n h y d r i n a f t e r s e p a r a t i o n b y i o n - e x c h a n g e c h r o m a t o g r a p h y w i t h a Dowex-5o c o l u m n ~. a - A l a n i n e w a s also d e t e r m i n e d m i c r o b i o l o g i c a l l y u s i n g L e u c o n o s t o c c i t r o v o r u m 8o8110 a n d t h e H e n d e r s o n - S n e l l m e d i u m 11 w h i c h c o n t a i n e d l e u c o v o r i n . (We a re i n d e b t e d t o Miss S. ISHIDA a n d Dr. T. SUZUKI for t h i s assay.)
THE STOICHIOMETRY OF THE R~ACTION
ko
0
0 B'I
to
voL. 33 (1959)
PRELIMINARY NOTES
593
The stoichiometry of the reaction is shown inTable I. a-Alanine was also identified by paper chromatography using five different solvent systems*. Malonic semialdehyde was isolated from a large-scale incubation mixture by ether extraction after acidification of the solution, and was identified as 2,4-dinitrophenylhydrazone by paper chromatography using three different solvent systems by comparison with an authentic sample**. Further evidence for the identity was provided by the quantitative decarboxylation with aniline citrate and by the production of equivalent amount of acetaldehyde upon steam distillation from the acidified reaction mixture. Acetaldehyde was also identified as the 2,4-dinitrophenylhydrazone. The reaction appears to be freely reversible, since the enzyme preparation catalyzes the stoichiometric formation of pyruvic acid and fl-alanine from a-alanine and malonic semialdehyde. Pyruvic acid acts as specific amino acceptor in the forward reaction. Neither a-ketoglutarate, fl-ketoglutarate, ~-ketoadipate nor oxaloacetate could replace pyruvate as amino acceptor. However, 7-aminobutyrate acts as amino donor to some extent. Preliminary evidence indicates that pyridoxal phosphate stimulates the activity of the dialyzed enzyme preparation. Studies are now in progress in order to elucidate the further metabolism of malonic semialdehyde. This investigation was supported in part by research grants from the National Institutes of Health (C-4222), the Rockefeller Foundation and the Jane Coffin Childs Memorial Fund for Medical Research. YASUTOMI N I S H I Z U K A
Depart~nent of Medical Cher~istry, Kyoto University Fa~ultv o/Medicine, Kyoto (Japan)
MASAZUMI T A K E S H I T A SIGERU
KUNO
OSAMU H A Y A I S H I 1 E. ROBERTS AND H. M. BREGOFF, J. Biol. Chem., 2Ol (1953) 393. 2 E. ROBERTS, P. AYENGER AND I. POSNER, J. Biol. Chem., '203 (1953) 195. 3 0 . HAYAISHI, M. KATAGIRI AND S. ROTHBERG, J. Biol. Chem., 229 (1957) 905. 4 p. G. WALKER, Biochem. J., 58 (1954) 699. s A. KORNBERG, J. Biol. Chem., 182 (195 o) 805. s N. L. EDSON, Biochem. J., 29 (1953) 2o82. E. EEGEIWE, Z. anal. Chem., 95 (1933) 323 • s E. RACKEE, J. Biol. Chem., 184 (195 o) 313 . 9 S. MOORE AND W. H. STEIN, ]. Biol. Chem., 176 (1948) 367 . 10 H . E. SAUBERLICH, J. Biol. Chem., 177 (1949) 177. 11 L. M. HENDERSON AND E. E. SNELL, J. Biol. Chem., 172 (1948) 15. 12 A. WOHL AND W . EMMERICH, Bet., 33 (19oo) 276o.
Received March 3Ist, 1959 * P a p e r c h r o m a t o g r a p h y w a s carried o u t on W h a t m a n No. i paper. Rp v a l u e s for a - a l a n i n e a n d t5-alanine w i t h i s o p r o p a n o l - w a t e r (8:2) were 0.33 a n d 0.25; w i t h e t h a n o l - a m m o n i a - w a t e r (18 :I :I) o.23 a n d o.I 1 ; w i t h n - b u t a n o l - p y r i d i n e - w a t e r (1:1:1) 0.43 a n d o.34; w i t h p h e n o l - w a t e r (8 :,2,) o.52 a n d 0.62; w i t h n - b u t a n o l - a c e t i c a c i d - w a t e r (4:2:3) 0.27 a n d o.33, respectively. T h e a u t h e n t i c s a m p l e w a s p r e p a r e d f r o m malonic s e m i a l d e h y d e w h i c h w a s s y n t h e s i z e d c h e m i c a l l y TM. W e are i n d e b t e d to Mr. H. KONDO, Drs. K. HAYASHI a n d T. SUZUKI for t h e chemical s y n t h e s i s of m a l o n i c s e m i a l d e h y d e . P a p e r c h r o m a t o g r a p h y w a s carried o u t on W h a t m a n No. I p a p e r a n d s p r a y e d w i t h dilute alkali. RF v a l u e s for t h e 2 , 4 - d i n i t r o p h e n y l h y d r a z o n e s of malonic s e m i a l d e h y d e a n d p y r u v i c acid w i t h i s o p r o p a n o l - i s o a m y l a l c o h o l - p y r i d i n e - w a t e r (2o: 4 :1:5) were 0.56 a n d 0.45; w i t h i s o a m y l a l c o h o l - e t h a n o l - o . i M c a r b o n a t e , p H lO. 7 (5:I:2, u p p e r layer) o.31 a n d o . i 3 ; w i t h n - b u t a n o l - e t h a n o l - o . I M c a r b o n a t e , p H lO. 7 (6:1 :i, lower layer) o.65 a n d o.56, respectively.