- Email: [email protected]
d -Glutamic acid and d -alanine as constituents of spores of Bacillus megatherium
VOL. 24 (1957)
SHO~T COMMUNICATIONS
199
D-Glutamic acid and D-alanine as constituents of spores of Bacillus rnegatherium Water-soluble, non-dialysable components ("spore peptide") were isolated from extracts of disi n t e g r a t e d resting spores a n d e x u d a t e s of germinating spores of Bacillus species1. After acid hydrolysis, t h e y contained ae-diaminopimelic acid, glutamic acid, alanine and acetyl derivatives of hexosamine and a s u b s t i t u t e d h e x o s a m i n e2,3. There is some evidence t h a t "spore peptide" is associated w i t h the coat of the resting spore 4, which is interesting in view of the presence of t h e same amino acids and amino sugars in acid hydrolysates of the isolated cell walls of a n u m b e r of vegetative, gram-positive organismss,e. The compositions of the isolated cell walls of Lactobacillus casei a n d Streptococcus taecalis h a v e been investigated recently b y SNELL, RADIN AND IgAWA7 and IKAWA AND S N E L L 8 who found t h a t considerat,le a m o u n t s of the total glutamic acid a n d alanine present in acid hydrolysates were the D-isomers. PARK9 reported the presence of b o t h D-alanine a n d D-glutamic acid together with amino sugars in a uridine-5'-phosphate comp o n e n t produced by penicillin-treated Staphylococcus aureus. I n view of these findings it was p e r t i n e n t to look for t h e presence of D-alanine a n d D-glutamic acid in "spore peptide". I n this investigation, B. megatherium "spore e x u d a t e peptide" was used; this, according to available physical criteria, is homogenous with a molecular weight of 15,3oo l°. The usual m e t h o d of e s t i m a t i n g D-glutamic acid h a s been t h e indirect one of subtracting the a m o u n t of t h e L-isomer, as determined with decarboxylase, from t h e total a m o u n t of glutamic acid. The recent finding by MIZUSHIMA et al. ix of a D-glutamic acid oxidase in AspergiUus u s t u s makes possible for t h e first time a convenient a n d accurate m e t h o d for t h e direct d e t e r m i n a t i o n of D-glutamic acid. T h e results reported here d e m o n s t r a t e the usefulness of this method, which was used in conjunction with t h e decarboxylase m e t h o d to obtain positive analyses of b o t h isomers. B . megatherium "spore peptide" (2oo rag) was hydrolysed b y heating in a sealed t u b e with 6 N HC1 (3 ml) for 2o h a n d t h e HC1 was removed b y distillation in vacuo. Glutamic acid a n d alanine were isolated from t h e hydrolysate by ion exchange c h r o m a t o g r a p h y . I n one procedure t h e h y d r o l y s a t e was passed t h r o u g h a column of Amberlite IR-I2O (H) a n d the amino acids were eluted with pyridine (lO% v/v). The pyridine was removed from the amino acids fraction b y distillation to dryness in vacuo and an aqueous solution of t h e residue was passed t h r o u g h a column of IR-4B (OH). Glutamic acid was eluted from the column with i N HC1. I n another procedure the amino acids were separated b y elution from a column of Dowex-5o(H ) with 0. 3 N HC1. Glutamic acid was partially freed from HC1 b y evaporation in vacuo b u t was n o t purified further. Alanine from t h e Dowex-5o column was freed from HC1 b y means of I R - 4 B (OH) and crystallized from ethanol-water. Total glutamic acid was determined b y paper c h r o m a t o g r a p h yTM. D-Glutamic acid was determined manometrically by m e a n s of D-glutamic acid oxidase from A s p . ustus 11. Fresh m y c e l i u m of A s p . ustus strain / was ground with a l u m i n a powder in o.o5M phosphate, p H 8.o, and treated as described by these authors. The volume of the final extract was reduced a b o u t 5 ° % by lyophilization. T h e preparation h a d little or no endogenous activity a n d remained highly active towards n - g l u t a m i c acid for 4 m o n t h s w h e n stored a t - - 2 o ° C. The preparation was completely inactive with L-glutamic acid, DL-alanine a n d all isomers of ae-diaminopimelic acid. The preparation (o.2 or o. 3 ml) was added to the side a r m of a W a r b u r g vessel and the sample at p H 8.0 was placed in the m a i n c o m p a r t m e n t . The final volume was m a d e up to 2, 3 ml with o.o5M p h o s p h a t e buffer, p H 8.o. L-Glutamic acid was determined manometrically with decarboxylase 13 b u t a lyophilized sonic e x t r a c t of Escherichia coli was used instead of dried cells. D-Alanine was determined manometrically with D-amino acid oxidase from sheep kidneys 14. No specific estimation of t h e L-isomer was m a d e b u t the alanine remaining after incubation with D-amino acid oxidase under conditions allowing the complete oxidation of the s a m e a m o u n t of D-alanine was e s t i m a t e d by paper c h r o m a t o g r a p h y with collidine-lutidine-HzO as solvent. Spots corresponding in position to alanine were located with ultraviolet light and c u t out a n d their amino acid c o n t e n t was determined. Q u a n t i t a t i v e assays of glutamic acid and alanine in hydrolysates are given in Table I. 99 9'o of the glutamic acid a n d 24 % of the alanine were the D-isomers. IKAWA AND SNELL8 reported t h a t hydrolysis of casein in 3 N HC1 in the presence of a n equal weight of starch racemized 12-2o% of the L-glutamic acid. B. megatherium "spore peptide" is rich in amino sugars b u t contains only a small a m o u n t of other carbohydrate 1. However, the possibility of racemization occurring during acid hydrolysis was checked b y heating m i x t u r e s of amino sugars, amino acids a n d glucose in acid, using a p p r o x i m a t e l y the concentrations found in hydrolysates of "spore peptide" (Table I). U n d e r these conditions little or no racemization occurred. A n a t t e m p t was m a d e to release glutamic acid from the peptide b y a n enzyme preparation from Bacillus subtilis which hydrolyses 7-D-glutamyl peptides 15. W h e n incubation m i x t u r e s of
200
SHORT COMMUNICATIONS
VOL. 24 (1057)
TABLE I ANALYSIS OF GLUTAMIC ACID AND ALANINE FROM ACID HYDROLYSATES OF "SPORE P E P T I D E " AND SIMULANT MIXTURES Sample
Peptide hydrolysate I
P e p t i d e h y d r o l y s a t e II
Mixture A Mixture B Mixture C D - G l u t a m i c acid L - G l u t a m i c acid
Amino add assayed
Total Itmole/ml
G l u t a m i c acid Alanine Alanine G l u t a m i c acid Alanine Alanine
4.2o 14.9o 7.58 4.53 33.4 ° 8.43
Glutamic Glutamic Alanine Glutamic Glutamic
4.80 13.6o 8.43 IO.O 5.0
acid acid acid acid
i~-Isomar L-Isomer pmole/ml % of total l*mole/ml % o] total
4.17 3.61
99.2 24.2
4.5I 7.95
99.6 23 .8
o
o
9.95
o.i2
~- *- LIsomers % o] total
2.9
io2.1
5.73* o.12
75 .6 2.6
99.8 lO2.2
6.69*
79.4
lO3.2
0.08 8.87*
0.6 lO5.2
4.99
99.8
99.5
P e p t i d e h y d r o l y s a t e I: P e p t i d e h y d r o l y s a t e II :
G l u t a m i c acid a n d a l a n i n e isolated on IR-I2O (H) a n d I R - 4 B (OH), G l u t a m i c acid a n d a l a n i n e isolated on D o w e x - 5 o. A l a n i n e crystallized from ethanol-water. M i x t u r e A: DL-alanine, 15 rag; g l u c o s a m i n e HC1, 5 ° m e ; ae-diaminopimelic acid, io rag; L-glutamic acid, IO m e ; glucose, 1. 5 m e ; acetic acid, 15 m e . M i x t u r e h e a t e d w i t h 4 m l of 6 N HC1 for 16 h a t lO5 °. M i x t u r e B: S a m e as A e x c e p t t h a t D - g l u t a m i c acid w a s used. M i x t u r e C: L-alanine, 2.1 rag; g l u c o s a m i n e HC1, 7 rng; a e - d i a m i n o p i m e l i c acid, 1. 4 rag; D - g l u t a m i c acid, 1. 5 rag; glucose, 0.6 rag. T r e a t e d w i t h HC1 as for A a n d B. * A l a n i n e r e m a i n i n g a f t e r t r e a t i n g w i t h D-amino acid oxidase. W h e n D-alanine w a s t r e a t e d u n d e r t h e s a m e c o n d i t i o n s , it w a s c o m p l e t e l y oxidized. t h i s e n z y m e a n d " s p o r e p e p t i d e " were a n a l y s e d , i n t e r e s t i n g r e s u l t s were o b t a i n e d . P a p e r chrom a t o g r a m s d e v e l o p e d w i t h p h e n o l - w a t e r s h o w e d a s p o t c o r r e s p o n d i n g in position to g l u t a m i c acid b u t t e s t s w i t h D - g l u t a m i c acid o x i d a s e failed to confirm t h e presence of D - g l u t a m i c acid in t h e i n c u b a t i o n m i x t u r e s . W h e n t h e s p o t w a s e l u t e d f r o m t h e paper, f u r t h e r i n v e s t i g a t i o n s h o w e d t h a t it w a s n o t free g l u t a m i c acid bu~ a p e p t i d e or m i x t u r e of p e p t i d e s c o n t a i n i n g g l u t a m i c acid, alanine, a e - d i a m i n o p i m e l i c acid a n d a m i n o sugars. A t least 4 o t h e r s p o t s were o b t a i n e d on p a p e r c h r o m a t o g r a m s of t h e s e e n z y m i c h y d r o l y s a t e s a n d p r e l i m i n a r y i n v e s t i g a t i o n s i n d i c a t e t h a t t h e p e p t i d a s e will be a useful tool for t h e release of s m a l l m o l e c u l a r w e i g h t peptides. T h e s t u d y of t h e s e will help in t h e e l u c i d a t i o n of t h e s t r u c t u r e of " s p o r e p e p t i d e " . W e are g r a t e f u l to Dr. K. SAKAGUCHI for s u p p l y i n g u s w i t h a c u l t u r e of Aspergillus ustus s t r a i n / .
Microbiological Research Establishment, Porton, Wiltshire (England)
R . E . STRANGE CURTIS B . T H O R N E *
1 R. E. STRANGE AND J. F. POWELL, Biochem. J., 58 (1954) 80. 2 R . E . STRANGE AND F. A . DARK, Nature, 177 (1956) 186. a R. E. STRANGE, Biochem. J., 64 (1956) 2 3 P . 4 R . E . STRANGE AND F . A . DARK, Biochem. J., 62 (1956) 4595 C. S. CUMMINS AND H . HARRIS, B i o c h e m . J., 5 7 (1954) x x x i i . 6 C. S. CUMMINS AND H . HARRIS, J . Gen. Microbiol., 14 (1956) 5 8 3 . ? E . E . SNELL, N . S. RADIN AND M. IKAWA, J. B i o l . C h e m . , 217 (1955) 8 0 3 . 8 M. IKAWA AND E. E. SNELL, Biochim. Biophys. Acta, 19 (1956) 576.
9 j . T. PARK, J. Biol. Chem., 194 (1952) 897. 10 B. R. RECORD AND K. H. GRINSTEAD, Biochem. J., 58 (1954) 85. iI S. MIZUSHIMA, K. IZAKI, H. TAKAHASHI AND K. SAKAGUCHI, Bull. Agr. Chem. Soc. Japan, 20 (1956) 36 • 18 R. D. HOOSEWRIGHT AND C. B. THORNE, J. Bacteriol., 60 (195o) 89. la W . W. UMBREIT AND I. C. GUNSALUS, J. Biol. Chem., 159 (1945) 333. 14 H. A. KREBS, Biochem. J., 29 (1935) I62O. 15 W. J. WILLIAMS, J. LITWlN AND C. B. THORNE, J. Biol. Chem., 212 (1955) 427 • R e c e i v e d J a n u a r y 4th, 1957 * D u r i n g a y e a r ' s w o r k i n g visit f r o m F o r t Derrick, Frederick, M a r y l a n d , U.S.A.
SHO~T COMMUNICATIONS
199
D-Glutamic acid and D-alanine as constituents of spores of Bacillus rnegatherium Water-soluble, non-dialysable components ("spore peptide") were isolated from extracts of disi n t e g r a t e d resting spores a n d e x u d a t e s of germinating spores of Bacillus species1. After acid hydrolysis, t h e y contained ae-diaminopimelic acid, glutamic acid, alanine and acetyl derivatives of hexosamine and a s u b s t i t u t e d h e x o s a m i n e2,3. There is some evidence t h a t "spore peptide" is associated w i t h the coat of the resting spore 4, which is interesting in view of the presence of t h e same amino acids and amino sugars in acid hydrolysates of the isolated cell walls of a n u m b e r of vegetative, gram-positive organismss,e. The compositions of the isolated cell walls of Lactobacillus casei a n d Streptococcus taecalis h a v e been investigated recently b y SNELL, RADIN AND IgAWA7 and IKAWA AND S N E L L 8 who found t h a t considerat,le a m o u n t s of the total glutamic acid a n d alanine present in acid hydrolysates were the D-isomers. PARK9 reported the presence of b o t h D-alanine a n d D-glutamic acid together with amino sugars in a uridine-5'-phosphate comp o n e n t produced by penicillin-treated Staphylococcus aureus. I n view of these findings it was p e r t i n e n t to look for t h e presence of D-alanine a n d D-glutamic acid in "spore peptide". I n this investigation, B. megatherium "spore e x u d a t e peptide" was used; this, according to available physical criteria, is homogenous with a molecular weight of 15,3oo l°. The usual m e t h o d of e s t i m a t i n g D-glutamic acid h a s been t h e indirect one of subtracting the a m o u n t of t h e L-isomer, as determined with decarboxylase, from t h e total a m o u n t of glutamic acid. The recent finding by MIZUSHIMA et al. ix of a D-glutamic acid oxidase in AspergiUus u s t u s makes possible for t h e first time a convenient a n d accurate m e t h o d for t h e direct d e t e r m i n a t i o n of D-glutamic acid. T h e results reported here d e m o n s t r a t e the usefulness of this method, which was used in conjunction with t h e decarboxylase m e t h o d to obtain positive analyses of b o t h isomers. B . megatherium "spore peptide" (2oo rag) was hydrolysed b y heating in a sealed t u b e with 6 N HC1 (3 ml) for 2o h a n d t h e HC1 was removed b y distillation in vacuo. Glutamic acid a n d alanine were isolated from t h e hydrolysate by ion exchange c h r o m a t o g r a p h y . I n one procedure t h e h y d r o l y s a t e was passed t h r o u g h a column of Amberlite IR-I2O (H) a n d the amino acids were eluted with pyridine (lO% v/v). The pyridine was removed from the amino acids fraction b y distillation to dryness in vacuo and an aqueous solution of t h e residue was passed t h r o u g h a column of IR-4B (OH). Glutamic acid was eluted from the column with i N HC1. I n another procedure the amino acids were separated b y elution from a column of Dowex-5o(H ) with 0. 3 N HC1. Glutamic acid was partially freed from HC1 b y evaporation in vacuo b u t was n o t purified further. Alanine from t h e Dowex-5o column was freed from HC1 b y means of I R - 4 B (OH) and crystallized from ethanol-water. Total glutamic acid was determined b y paper c h r o m a t o g r a p h yTM. D-Glutamic acid was determined manometrically by m e a n s of D-glutamic acid oxidase from A s p . ustus 11. Fresh m y c e l i u m of A s p . ustus strain / was ground with a l u m i n a powder in o.o5M phosphate, p H 8.o, and treated as described by these authors. The volume of the final extract was reduced a b o u t 5 ° % by lyophilization. T h e preparation h a d little or no endogenous activity a n d remained highly active towards n - g l u t a m i c acid for 4 m o n t h s w h e n stored a t - - 2 o ° C. The preparation was completely inactive with L-glutamic acid, DL-alanine a n d all isomers of ae-diaminopimelic acid. The preparation (o.2 or o. 3 ml) was added to the side a r m of a W a r b u r g vessel and the sample at p H 8.0 was placed in the m a i n c o m p a r t m e n t . The final volume was m a d e up to 2, 3 ml with o.o5M p h o s p h a t e buffer, p H 8.o. L-Glutamic acid was determined manometrically with decarboxylase 13 b u t a lyophilized sonic e x t r a c t of Escherichia coli was used instead of dried cells. D-Alanine was determined manometrically with D-amino acid oxidase from sheep kidneys 14. No specific estimation of t h e L-isomer was m a d e b u t the alanine remaining after incubation with D-amino acid oxidase under conditions allowing the complete oxidation of the s a m e a m o u n t of D-alanine was e s t i m a t e d by paper c h r o m a t o g r a p h y with collidine-lutidine-HzO as solvent. Spots corresponding in position to alanine were located with ultraviolet light and c u t out a n d their amino acid c o n t e n t was determined. Q u a n t i t a t i v e assays of glutamic acid and alanine in hydrolysates are given in Table I. 99 9'o of the glutamic acid a n d 24 % of the alanine were the D-isomers. IKAWA AND SNELL8 reported t h a t hydrolysis of casein in 3 N HC1 in the presence of a n equal weight of starch racemized 12-2o% of the L-glutamic acid. B. megatherium "spore peptide" is rich in amino sugars b u t contains only a small a m o u n t of other carbohydrate 1. However, the possibility of racemization occurring during acid hydrolysis was checked b y heating m i x t u r e s of amino sugars, amino acids a n d glucose in acid, using a p p r o x i m a t e l y the concentrations found in hydrolysates of "spore peptide" (Table I). U n d e r these conditions little or no racemization occurred. A n a t t e m p t was m a d e to release glutamic acid from the peptide b y a n enzyme preparation from Bacillus subtilis which hydrolyses 7-D-glutamyl peptides 15. W h e n incubation m i x t u r e s of
200
SHORT COMMUNICATIONS
VOL. 24 (1057)
TABLE I ANALYSIS OF GLUTAMIC ACID AND ALANINE FROM ACID HYDROLYSATES OF "SPORE P E P T I D E " AND SIMULANT MIXTURES Sample
Peptide hydrolysate I
P e p t i d e h y d r o l y s a t e II
Mixture A Mixture B Mixture C D - G l u t a m i c acid L - G l u t a m i c acid
Amino add assayed
Total Itmole/ml
G l u t a m i c acid Alanine Alanine G l u t a m i c acid Alanine Alanine
4.2o 14.9o 7.58 4.53 33.4 ° 8.43
Glutamic Glutamic Alanine Glutamic Glutamic
4.80 13.6o 8.43 IO.O 5.0
acid acid acid acid
i~-Isomar L-Isomer pmole/ml % of total l*mole/ml % o] total
4.17 3.61
99.2 24.2
4.5I 7.95
99.6 23 .8
o
o
9.95
o.i2
~- *- LIsomers % o] total
2.9
io2.1
5.73* o.12
75 .6 2.6
99.8 lO2.2
6.69*
79.4
lO3.2
0.08 8.87*
0.6 lO5.2
4.99
99.8
99.5
P e p t i d e h y d r o l y s a t e I: P e p t i d e h y d r o l y s a t e II :
G l u t a m i c acid a n d a l a n i n e isolated on IR-I2O (H) a n d I R - 4 B (OH), G l u t a m i c acid a n d a l a n i n e isolated on D o w e x - 5 o. A l a n i n e crystallized from ethanol-water. M i x t u r e A: DL-alanine, 15 rag; g l u c o s a m i n e HC1, 5 ° m e ; ae-diaminopimelic acid, io rag; L-glutamic acid, IO m e ; glucose, 1. 5 m e ; acetic acid, 15 m e . M i x t u r e h e a t e d w i t h 4 m l of 6 N HC1 for 16 h a t lO5 °. M i x t u r e B: S a m e as A e x c e p t t h a t D - g l u t a m i c acid w a s used. M i x t u r e C: L-alanine, 2.1 rag; g l u c o s a m i n e HC1, 7 rng; a e - d i a m i n o p i m e l i c acid, 1. 4 rag; D - g l u t a m i c acid, 1. 5 rag; glucose, 0.6 rag. T r e a t e d w i t h HC1 as for A a n d B. * A l a n i n e r e m a i n i n g a f t e r t r e a t i n g w i t h D-amino acid oxidase. W h e n D-alanine w a s t r e a t e d u n d e r t h e s a m e c o n d i t i o n s , it w a s c o m p l e t e l y oxidized. t h i s e n z y m e a n d " s p o r e p e p t i d e " were a n a l y s e d , i n t e r e s t i n g r e s u l t s were o b t a i n e d . P a p e r chrom a t o g r a m s d e v e l o p e d w i t h p h e n o l - w a t e r s h o w e d a s p o t c o r r e s p o n d i n g in position to g l u t a m i c acid b u t t e s t s w i t h D - g l u t a m i c acid o x i d a s e failed to confirm t h e presence of D - g l u t a m i c acid in t h e i n c u b a t i o n m i x t u r e s . W h e n t h e s p o t w a s e l u t e d f r o m t h e paper, f u r t h e r i n v e s t i g a t i o n s h o w e d t h a t it w a s n o t free g l u t a m i c acid bu~ a p e p t i d e or m i x t u r e of p e p t i d e s c o n t a i n i n g g l u t a m i c acid, alanine, a e - d i a m i n o p i m e l i c acid a n d a m i n o sugars. A t least 4 o t h e r s p o t s were o b t a i n e d on p a p e r c h r o m a t o g r a m s of t h e s e e n z y m i c h y d r o l y s a t e s a n d p r e l i m i n a r y i n v e s t i g a t i o n s i n d i c a t e t h a t t h e p e p t i d a s e will be a useful tool for t h e release of s m a l l m o l e c u l a r w e i g h t peptides. T h e s t u d y of t h e s e will help in t h e e l u c i d a t i o n of t h e s t r u c t u r e of " s p o r e p e p t i d e " . W e are g r a t e f u l to Dr. K. SAKAGUCHI for s u p p l y i n g u s w i t h a c u l t u r e of Aspergillus ustus s t r a i n / .
Microbiological Research Establishment, Porton, Wiltshire (England)
R . E . STRANGE CURTIS B . T H O R N E *
1 R. E. STRANGE AND J. F. POWELL, Biochem. J., 58 (1954) 80. 2 R . E . STRANGE AND F. A . DARK, Nature, 177 (1956) 186. a R. E. STRANGE, Biochem. J., 64 (1956) 2 3 P . 4 R . E . STRANGE AND F . A . DARK, Biochem. J., 62 (1956) 4595 C. S. CUMMINS AND H . HARRIS, B i o c h e m . J., 5 7 (1954) x x x i i . 6 C. S. CUMMINS AND H . HARRIS, J . Gen. Microbiol., 14 (1956) 5 8 3 . ? E . E . SNELL, N . S. RADIN AND M. IKAWA, J. B i o l . C h e m . , 217 (1955) 8 0 3 . 8 M. IKAWA AND E. E. SNELL, Biochim. Biophys. Acta, 19 (1956) 576.
9 j . T. PARK, J. Biol. Chem., 194 (1952) 897. 10 B. R. RECORD AND K. H. GRINSTEAD, Biochem. J., 58 (1954) 85. iI S. MIZUSHIMA, K. IZAKI, H. TAKAHASHI AND K. SAKAGUCHI, Bull. Agr. Chem. Soc. Japan, 20 (1956) 36 • 18 R. D. HOOSEWRIGHT AND C. B. THORNE, J. Bacteriol., 60 (195o) 89. la W . W. UMBREIT AND I. C. GUNSALUS, J. Biol. Chem., 159 (1945) 333. 14 H. A. KREBS, Biochem. J., 29 (1935) I62O. 15 W. J. WILLIAMS, J. LITWlN AND C. B. THORNE, J. Biol. Chem., 212 (1955) 427 • R e c e i v e d J a n u a r y 4th, 1957 * D u r i n g a y e a r ' s w o r k i n g visit f r o m F o r t Derrick, Frederick, M a r y l a n d , U.S.A.