- Email: [email protected]
The inhibition of Escherichia coli by l -penicillamine and its reversal by isoleucine, valine or leucine
2i 4
PRELZMINARY
NOTES
VOL 30 (z958)
The inhibition of Escherichm coli by L-penicillamine and its reversal by isoleucine, valine or leucine* The sulfhydryl amino acid L-penicillamine inhibits the growth of ~ats 1 a n d the acid production of Leuconostoc mesenteroides P-6o 2,8. D u VIGNEAUD and co-workers 1, 4 h a v e shown t h a t in the r a t t h e growth-inhibitory a c t i v i t y is reversed b y either aminoethanol, its N - m e t h y l a t e d derivatives or pyridoxine. APOSHIAN and co-workers2, 3 h a v e reported t h a t L-serine reverses the inhibitory a c t i v i t y of this sulfhydryl amino acid in Leuconos~oc mesenteroides P-6o. The experiments of the present report indicate t h a t L-penicillamine inhibits the growth of Escherichia coli and t h a t the inhibition is completely reversed b y a n y one of three n a t u r a l l y occurring amino acids t h a t are analogous in chemical s t r u c t u r e to L-penicillamine. 0.300-
Jq
0.200-
0.100-
o
,.o
2'.o
J4 MOLES O r
3:o L-PEN
41o PER
slo
d.o
ML
Fig. I. Inhibition of t h e IT-h growth of 2£. coli No. 9723 b y L-penicillamine. Growth was measured with a n E v e l y n photoelectric colorimeter (65o filter) using distilled water to a d j u s t the i n s t r u m e n t to ioo % light transmission. The g a l v a n o m e t e r readings were converted to photometric density, a n expression of culture turbidity.
As s h o w n in Fig. I, L-peniciUamine in a concentration of i.I /~moles/ml m e d i u m produced 5 ° % inhibition of t h e m a x i m u m growth of E. coli A.T.C.C. No. 9723 w h e n this organism was grown for 17 h a t 37 ° in DUNN'S ~ modification of ANDERSON'S6 medium, containing salts, NH4C1 and glucose. As in the ratX~ D-penicillamine is devoid of such growth-inhibitory activity for this microorganism. Either 13-isoleucine, L-valine, L-leucine or DL-methionine was found completely to reverse t h e growth-inhibitory activity of L-penicillamine. Table I presents t h e m i n i m u m a m o u n t of these a m i n o acids necessary for reversal. W h e n glutathione or a n y of 16 n a t u r a l l y occurring a m i n o acids, including serine, were added individually in concentrations up to i6 /,moleslml m e d i u m , t h e growth-inhibitory effects of 4 /~moles L-penicillamine]ml were n o t reversed in this organism. W h e n aminoethanol, choline, pyridoxine, or pyridoxamine was added in concentration u p to 16/zmoles/ml m e d i u m , it was found t h a t t h e inhibitory activity of 4 #,moles L-penicillamine/ml m e d i u m was n o t reversed. Since aminoethanol, choline or pyridoxine will reverse t h e rat-growth* T h i s s t u d y was s u p p o r t e d in p a r t b y t h e National Cancer I n s t i t u t e (Grant No. C-2572) a n d b y t h e W i l l i a m s - W a t e r m a n F u n d for t h e C o m b a t of Dietary Diseases.
VOL. 30 (1958)
PRELIMINARY NOTES
215
TABLE I MINIMUM AMOUNTS OF AMINO ACIDS THAT IBIDIV~DUALLYAND COMPLETELY PJgVRRSE THE GROWTH-INHIBITORY ACTIVITY FOR E. coli OF 4 ~MOLES L-PENICILLAMINE/ML MBDIUM I n c u b a t i o n 17 h a t 37°; E . coli No. 9723. A mino A c/d
L-Isoleucine L-Valine L-Leucine DL-Methionine
#mo/e~Im/
o. I o o.2o 0.40 8.o
i n h i b i t o r y a c t i v i t y of t h i s s u l f h y d r y l a m i n o acidl, 4, it a p p e a r s t h a t t h e m o d e of action of Lp e n i c i l i a m i n e in t h e r a t a n d in E. coli differ. I t h a s b e e n s u g g e s t e d t h a t t h e f o r m a t i o n of t h e thiazolidine of p y r i d o x a l - 5 - p h o s p h a t e a n d L-penicillamine m a y p l a y a role in t h e g r o w t h - i n h i b i t o r y a c t i v i t y of L-penicillamine in t h e r a t b y i n d u c i n g a v i t a m i n B 6 deficiency% s. I t w a s n o t possible to o b t a i n e x p e r i m e n t a l evidence for s u c h a m e c h a n i s m in E. coli since p y r i d o x a l or its p h o s p h a t e in c o n c e n t r a t i o n s of 0.8 or 4 . o / ~ m o l e s ] m l m e d i u m r e s p e c t i v e l y is i n h i b i t o r y per se. L o w e r a n d m o r e physiological a m o u n t s of t h e s e t w o m e m b e r s of t h e v i t a m i n B e c o m p l e x do n o t reverse t h e i n h i b i t o r y a c t i v i t y of 4 /*moles L-peniciUamine[ml m e d i u m . I t is of i n t e r e s t to n o t e t h a t it h a s been s h o w n t h a t L-penicillamine is o n l y s l i g h t l y i n h i b i t o r y to t h e utilization of t h e v i t a m i n B s c o m p l e x b y S. carlsbergensis, a v i t a m i n Be-requiring o r g a n i s m g. Since either isoleucine, valine or leucine, a m i n o acids a n a l o g o u s in chemical s t r u c t u r e to L-peniciUamine, c o m p l e t e l y reverses t h e g r o w t h - i n h i b i t o r y a c t i v i t y of this s u l f h y d r y l a m i n o acid in E. coli, it a p p e a r s u n l i k e l y t h a t L-penicillamine is i n d u c i n g a generalized v i t a m i n B, deficiency in t h i s o r g a n i s m . If s u c h were so, o t h e r a m i n o acids w h i c h serve as s u b s t r a t e s or p r o d u c t s of v i t a m i n - B e - r e q u i r i n g e n z y m e r e a c t i o n s w o u l d be expected to reverse t h e inhibition to s o m e e x t e n t a n d it w o u l d n o t be e x p e c t e d t h a t a single a m i n o acid w o u l d p r o d u c e a c o m p l e t e reversal. R a t h e r , it a p p e a r s t h a t one or m o r e of t h e s e t h r e e a m i n o acids is c o m p e t i n g w i t h penicillamine for a specific p r o t e i n or e n z y m e in E. coli.
Department o/Pharmacology, Vanderbilt University School o] Medicine, Nashville 5, Tenn. (U.S.A.)
ROBERT M. BLAIR H. VASKEN APOSHIAN
I j . E. WILSON AND V. DU VIGNEAUD, J. Biol. Chem., 184 (195o) 63. g H . V. APOSHIAN AND J. A. SETLIFF, Federation Proc., 15 (1956) 212. s H. V. APOSHIAN, W. G. RHEA AND S. M. WOLFF, Arch. Biochem. Biophys., 71 (1957) 442. 4 E. J. KUCHINSHAS AND V. DU VIGNEAUD, Arch. Biochem. Biophys., 66 (1957) I. 5 F. W. DUNN, J. IV[. RAVEL AND W . SHIVE, J. Biol. Chem., 219 (1956) 809. 6 E. H. ANDERSON, Proc. Natl. Acad. Sci. U.S., 32 (1946) I2O. 7 E. J. KUCHINSKAS, A. HORVATH AND V. DU VIGNEAUD, Arch. Biochem. Biophys., 68 (1957) 69. 8 V . DU VIGNEAUD, E . J. KUCHINSKAS AND A. HORVA~:H, Arch. Biochem. Biophys., 69 (I957) 13o. 9 H. V. APOSHIAN, M. MORRIS AND M. M. APOSHIAN, Abstraas, I32nd Meeting Am. Chem. Sot., I957, p. 93c. Received J u n e 25th, 1958
Depolymerization of deoxyribonucleicacid by the juice of soy-bean sprouts D e o x y r i b o n u c l e a s e s of h i g h e r p l a n t s h a v e n o t been r e p o r t e d e x c e p t for t h a t of g e r m i n a t i n g b a r l e y 1 a n d of o n i o n 2. A r e m a r k a b l e deoxyribonuclease a c t i v i t y was f o u n d in t h e juice of s p r o u t s of Phaseolus radiatus var. t y p i c u s . T h i s d e o x y r i b o n u c l e a s e w a s p a r t i a l l y purified b y s a l t i n g - o u t w i t h a m m o n i u m sulfate. F r e s h s o y - b e a n s p r o u t s were w a s h e d w i t h w a t e r a n d h o m o g e n i z e d w i t h a W a r i n g B l e n d o r a t 3,6oo r e v / m i n for 5 m l n in t h e cold. T h e h o m o g e n a t e w a s filtered w i t h t h e aid of S t a n d a r d Super Cel (SSC) a n d c o n c e n t r a t e d to a b o u t o n e - t h i r d vol. a t a t e m p e r a t u r e lower t h a n 3o ° a n d t h e r e s u l t a n t p r e c i p i t a t e s discarded. A f t e r t h e e l i m i n a t i o n of t h e precipitate f o r m e d b y a 6o % s a t u r a t i o n w i t h a m m o n i u m sulfate, t h e s u p e r n a t a n t w a s b r o u g h t to a 8 0 % s a t u r a t i o n . T h e p r e c i p i t a t e w a s
PRELZMINARY
NOTES
VOL 30 (z958)
The inhibition of Escherichm coli by L-penicillamine and its reversal by isoleucine, valine or leucine* The sulfhydryl amino acid L-penicillamine inhibits the growth of ~ats 1 a n d the acid production of Leuconostoc mesenteroides P-6o 2,8. D u VIGNEAUD and co-workers 1, 4 h a v e shown t h a t in the r a t t h e growth-inhibitory a c t i v i t y is reversed b y either aminoethanol, its N - m e t h y l a t e d derivatives or pyridoxine. APOSHIAN and co-workers2, 3 h a v e reported t h a t L-serine reverses the inhibitory a c t i v i t y of this sulfhydryl amino acid in Leuconos~oc mesenteroides P-6o. The experiments of the present report indicate t h a t L-penicillamine inhibits the growth of Escherichia coli and t h a t the inhibition is completely reversed b y a n y one of three n a t u r a l l y occurring amino acids t h a t are analogous in chemical s t r u c t u r e to L-penicillamine. 0.300-
Jq
0.200-
0.100-
o
,.o
2'.o
J4 MOLES O r
3:o L-PEN
41o PER
slo
d.o
ML
Fig. I. Inhibition of t h e IT-h growth of 2£. coli No. 9723 b y L-penicillamine. Growth was measured with a n E v e l y n photoelectric colorimeter (65o filter) using distilled water to a d j u s t the i n s t r u m e n t to ioo % light transmission. The g a l v a n o m e t e r readings were converted to photometric density, a n expression of culture turbidity.
As s h o w n in Fig. I, L-peniciUamine in a concentration of i.I /~moles/ml m e d i u m produced 5 ° % inhibition of t h e m a x i m u m growth of E. coli A.T.C.C. No. 9723 w h e n this organism was grown for 17 h a t 37 ° in DUNN'S ~ modification of ANDERSON'S6 medium, containing salts, NH4C1 and glucose. As in the ratX~ D-penicillamine is devoid of such growth-inhibitory activity for this microorganism. Either 13-isoleucine, L-valine, L-leucine or DL-methionine was found completely to reverse t h e growth-inhibitory activity of L-penicillamine. Table I presents t h e m i n i m u m a m o u n t of these a m i n o acids necessary for reversal. W h e n glutathione or a n y of 16 n a t u r a l l y occurring a m i n o acids, including serine, were added individually in concentrations up to i6 /,moleslml m e d i u m , t h e growth-inhibitory effects of 4 /~moles L-penicillamine]ml were n o t reversed in this organism. W h e n aminoethanol, choline, pyridoxine, or pyridoxamine was added in concentration u p to 16/zmoles/ml m e d i u m , it was found t h a t t h e inhibitory activity of 4 #,moles L-penicillamine/ml m e d i u m was n o t reversed. Since aminoethanol, choline or pyridoxine will reverse t h e rat-growth* T h i s s t u d y was s u p p o r t e d in p a r t b y t h e National Cancer I n s t i t u t e (Grant No. C-2572) a n d b y t h e W i l l i a m s - W a t e r m a n F u n d for t h e C o m b a t of Dietary Diseases.
VOL. 30 (1958)
PRELIMINARY NOTES
215
TABLE I MINIMUM AMOUNTS OF AMINO ACIDS THAT IBIDIV~DUALLYAND COMPLETELY PJgVRRSE THE GROWTH-INHIBITORY ACTIVITY FOR E. coli OF 4 ~MOLES L-PENICILLAMINE/ML MBDIUM I n c u b a t i o n 17 h a t 37°; E . coli No. 9723. A mino A c/d
L-Isoleucine L-Valine L-Leucine DL-Methionine
#mo/e~Im/
o. I o o.2o 0.40 8.o
i n h i b i t o r y a c t i v i t y of t h i s s u l f h y d r y l a m i n o acidl, 4, it a p p e a r s t h a t t h e m o d e of action of Lp e n i c i l i a m i n e in t h e r a t a n d in E. coli differ. I t h a s b e e n s u g g e s t e d t h a t t h e f o r m a t i o n of t h e thiazolidine of p y r i d o x a l - 5 - p h o s p h a t e a n d L-penicillamine m a y p l a y a role in t h e g r o w t h - i n h i b i t o r y a c t i v i t y of L-penicillamine in t h e r a t b y i n d u c i n g a v i t a m i n B 6 deficiency% s. I t w a s n o t possible to o b t a i n e x p e r i m e n t a l evidence for s u c h a m e c h a n i s m in E. coli since p y r i d o x a l or its p h o s p h a t e in c o n c e n t r a t i o n s of 0.8 or 4 . o / ~ m o l e s ] m l m e d i u m r e s p e c t i v e l y is i n h i b i t o r y per se. L o w e r a n d m o r e physiological a m o u n t s of t h e s e t w o m e m b e r s of t h e v i t a m i n B e c o m p l e x do n o t reverse t h e i n h i b i t o r y a c t i v i t y of 4 /*moles L-peniciUamine[ml m e d i u m . I t is of i n t e r e s t to n o t e t h a t it h a s been s h o w n t h a t L-penicillamine is o n l y s l i g h t l y i n h i b i t o r y to t h e utilization of t h e v i t a m i n B s c o m p l e x b y S. carlsbergensis, a v i t a m i n Be-requiring o r g a n i s m g. Since either isoleucine, valine or leucine, a m i n o acids a n a l o g o u s in chemical s t r u c t u r e to L-peniciUamine, c o m p l e t e l y reverses t h e g r o w t h - i n h i b i t o r y a c t i v i t y of this s u l f h y d r y l a m i n o acid in E. coli, it a p p e a r s u n l i k e l y t h a t L-penicillamine is i n d u c i n g a generalized v i t a m i n B, deficiency in t h i s o r g a n i s m . If s u c h were so, o t h e r a m i n o acids w h i c h serve as s u b s t r a t e s or p r o d u c t s of v i t a m i n - B e - r e q u i r i n g e n z y m e r e a c t i o n s w o u l d be expected to reverse t h e inhibition to s o m e e x t e n t a n d it w o u l d n o t be e x p e c t e d t h a t a single a m i n o acid w o u l d p r o d u c e a c o m p l e t e reversal. R a t h e r , it a p p e a r s t h a t one or m o r e of t h e s e t h r e e a m i n o acids is c o m p e t i n g w i t h penicillamine for a specific p r o t e i n or e n z y m e in E. coli.
Department o/Pharmacology, Vanderbilt University School o] Medicine, Nashville 5, Tenn. (U.S.A.)
ROBERT M. BLAIR H. VASKEN APOSHIAN
I j . E. WILSON AND V. DU VIGNEAUD, J. Biol. Chem., 184 (195o) 63. g H . V. APOSHIAN AND J. A. SETLIFF, Federation Proc., 15 (1956) 212. s H. V. APOSHIAN, W. G. RHEA AND S. M. WOLFF, Arch. Biochem. Biophys., 71 (1957) 442. 4 E. J. KUCHINSHAS AND V. DU VIGNEAUD, Arch. Biochem. Biophys., 66 (1957) I. 5 F. W. DUNN, J. IV[. RAVEL AND W . SHIVE, J. Biol. Chem., 219 (1956) 809. 6 E. H. ANDERSON, Proc. Natl. Acad. Sci. U.S., 32 (1946) I2O. 7 E. J. KUCHINSKAS, A. HORVATH AND V. DU VIGNEAUD, Arch. Biochem. Biophys., 68 (1957) 69. 8 V . DU VIGNEAUD, E . J. KUCHINSKAS AND A. HORVA~:H, Arch. Biochem. Biophys., 69 (I957) 13o. 9 H. V. APOSHIAN, M. MORRIS AND M. M. APOSHIAN, Abstraas, I32nd Meeting Am. Chem. Sot., I957, p. 93c. Received J u n e 25th, 1958
Depolymerization of deoxyribonucleicacid by the juice of soy-bean sprouts D e o x y r i b o n u c l e a s e s of h i g h e r p l a n t s h a v e n o t been r e p o r t e d e x c e p t for t h a t of g e r m i n a t i n g b a r l e y 1 a n d of o n i o n 2. A r e m a r k a b l e deoxyribonuclease a c t i v i t y was f o u n d in t h e juice of s p r o u t s of Phaseolus radiatus var. t y p i c u s . T h i s d e o x y r i b o n u c l e a s e w a s p a r t i a l l y purified b y s a l t i n g - o u t w i t h a m m o n i u m sulfate. F r e s h s o y - b e a n s p r o u t s were w a s h e d w i t h w a t e r a n d h o m o g e n i z e d w i t h a W a r i n g B l e n d o r a t 3,6oo r e v / m i n for 5 m l n in t h e cold. T h e h o m o g e n a t e w a s filtered w i t h t h e aid of S t a n d a r d Super Cel (SSC) a n d c o n c e n t r a t e d to a b o u t o n e - t h i r d vol. a t a t e m p e r a t u r e lower t h a n 3o ° a n d t h e r e s u l t a n t p r e c i p i t a t e s discarded. A f t e r t h e e l i m i n a t i o n of t h e precipitate f o r m e d b y a 6o % s a t u r a t i o n w i t h a m m o n i u m sulfate, t h e s u p e r n a t a n t w a s b r o u g h t to a 8 0 % s a t u r a t i o n . T h e p r e c i p i t a t e w a s