15.2. 1975
Specialia
t h e n again, it m a y be t h a t t h e i n t r a n u c l e a r calcium t a k e s p a r t in i n t r a c e l l u l a r c a l c i u m kinetics. W h e t h e r t h e c h a n g e s are r e l a t e d t o m e c h a n i c a l muscle a c t i v i t y or are an e x p r e s s i o n of a d a p t a t i o n processes r e m a i n s unclear.
157
Rdsumd. L ' a n a l y s e p a r m i c r o s o n d e 61ectronique des cellules m u s c u l a i r e s m o n t r e de h a u t e s c o n c e n t r a t i o n s de c a l c i u m d a n s tes n o y a n x ; la c o n c e n t r a t i o n p e u t fitre modifi6e p h a r m a c o l o g i q u e m e n t ou p a r , p a c e m a k e r ~ 61ectrique R. YAROM, T. A. HALL a n d P. D. PETERS 15
15 We acknowledge the technical assistance of M. ScRiePs and S. SCHWARTZ.We thank B. GOLEKfor editorial help. This research was supported by the British Science Research Council, the Joint Research Fund of the Hebrew University-Hadassah Medical School, and an EMBO travelling fellowship for R.Y.
Department o/Pathology, Hebrew University-Hadassah Medical School, P.O. Box 7772, Jerusalem (Israel), and University o/ Cambridge, Cavendish Laboratory, Free School Lane, Cambridge CB2 3RQ ~England)s 27 May 7974.
Organophosphate-Detoxicating Enzymes in E. coli. Gelfiltration and Isoelectric Focusing of DFPase, Paraoxonase and Unspecific Phosphohydrolases Toxic o r g a n 0 p h o s p h a t e s , c o m m o n l y k n o w n as insecticides or as n e r v e gases, are i n h i b i t o r s of cholinesterases, thereby blocking nerve translnissionL Some hydrolytic e n z y m e s ( ' p h o s p h o r y l p h o s p h a t a s e s ' ) are c a p a b l e of s p l i t t i n g t h e a c i d a n h y d r i d e b o n d of o r g a n o p h o s p h a t e poisons, f o r m i n g n o n - t o x i c o r g a n o p h o s p h o r u s acid2, ~. The following p a p e r describes t h e e n z y m a t i c organop h o s p h a t e d e t o x i c a t i o n b y E. coli p h o s p h o h y d r o l a s e s . Two d e t o x i c a t i n g e n z y m e s were f m m d , a D F P a s e (E.C. 3.8.2.1) a n d a p a r a o x o n a s e (arylesterase, E.C. 3.1.1.2). D F P a s e - a c t i v i t y was d e t e r m i n e d b y 3 m e t h o d s w i t h d i - i s o p r o p y l f l u o r o p h o s p h a t e ( D F P ) as s u b s t r a t e : titrim e t r i c a l l y 4, m a n o m e t r i c a l l y 5 or b y m e a s u r i n g t h e liber-
Fig. 1. Geliiltration of E. coli extract on Sephadex G-200 (2.5 • 73 cm). 4 mi extract (2 g wet ceils) containing 73 mg protein and 562 mU DFPase were fraetionated at I~ flow rate: 0.2 ml/min. O, protein (E 1260J; emx 9 DFPase (mU/ml).
a t e d fluoride w i t h an i o n s e n s i t i v e electrode ~. P a r a o x o n a s e was t e s t e d colorimetrically w i t h 0 , 0 - d i e t h y l - 0 - p - n i t r o p h e n y l p h o s p h a t e (paraoxon)7. Alkaline a n d acid p h o s p h a t a s e were m e a s u r e d colorimetrically w i t h p - n i t r o p h e n y l p h o s p h a t e in 0.2 M Tris-buffer a t p H 9 a n d p i t 6, t h e a b s o r b a n c e a t 405 n m was r e a d a f t e r d i l u t i n g s a m p l e s with 1 M sodium hydroxide. Phosphodiesterase determin a t i o n s were p e r f o r m e d w i t h bis-p-nitrophenylphosphate a t p H 8 in t h e s a m e way. All t e s t s were p e r f o r m e d a t 25 ~ a c t i v i t y is e x p r e s s e d as 1 a m o l e s u b s t r a t e h y d r o l y z e d per min. E. coli K12sr e x t r a c t s were p r e p a r e d according to MCILwAIN 8. T h e e x t r a c t was f r a c t i o n a t e d b y gelfiltratioI1 on a S e p h a d e x G-200 columnl t h e elution d i a g r a m for p r o t e i n u n d D F P a s e is s h o w n in Figure 1. The d i s t r i b u t i o n coefficients (Kd-valus) of t h e 2 D F P a s e p e a k s were c a l c u l a t e d 9 a n d c o m p a r e d w i t h t h o s e of Other p h o s p h o h y d r o l a s e s in a second e x p e r i m e n t on t h e s a m e c o l u m n (Table I). D F P a s e a n d p a r a o x o n a s e could clearly be s e p e r a t e d b y gelfiltration; no o v e r l a p p i n g a c t i v i t y was d e t e c t e d : D F P a s e f r a c t i o n s did n o t h y d r o l y z e p a r a o x o n a n d vice versa. T h e r e was no h y d r o l y s i s of p - n i t r o p h e n y l p h o s p h a t e at acid or alkaline p i t or of bis-p-nitro-phenylphosphate b y e i t h e r t h e D F P a s e or p a r a o x o n a s e c o n t a i n i n g fractions. The p h o s p h o h y d r o l a s e s in E. coli e x t r a c t s were a d d i t i o n a l l y s u b j e c t e d to free p r e p a r a t i v e isoelectric f o c u s i n g l 0 , n . F o u r D F P a s e p e a k s w i t h is0electric p o i n t s of 5.3, 5.7, 6.1 a n d 7.8 were f o u n d (Figure 2). T h e isoelectric p o i n t s of t h e o t h e r p h o s p h o h y d r o l a s e s were det e r m i n e d in a n i d e n t i c a l w a y in a d i f f e r e n t e x p e r i m e n t , t h e isoelectric p o i n t s are listed in Table II. The p h o s p h o h y d r o l a s e s of E. coli s h o w e d similar or e v e n identical isoelectric points, so, in c o n t r a s t t o t h e gelfiltration e x p e r i m e n t , t h e y could n o t be s e p a r a t e d b y t h i s m e t h o d . Coli D F P a s e h a s a low s u b s t r a t e a f f i n i t y ( K m 1.7 • 10 -~ M/1 for D F P ) a n d shows p u r e MICHAELIS-MENTEN kinetic (Figure 3). T h e p H - d e p e n d e n c e of coli D F P a s e is s h o w n
Table I. Distribution coefficients (Kd-valus) of E. coli phosphohydrolases oI~ Sephadex G-200 (2.5 • 73 cm)
Table II. Isoelectric points of E. coli phosphohydrolases determined by free preparative isoelectrie focusing in 2% ampholine, pH 3-6
Enzyme
Kd-valus
Enzyme
DFPase Paraoxonase Acid phosphatase Alkaline phosphatase Phosphodiesterase
0.21 0.11 0.39 0.42 0.40
10 ~2
uJ
&
.E 0
ro
1
0
I00
150
200
250
300
350 ml
Eluate
0.31 0.55
0.66
DFPase Paraoxonase Acid phosphatase Alkaline phosphatase Phosphodiesterase
Isoelectric points 5.3 5.3 4.9 4.8 5.0
5.7 5.6 5.3 5.3 5.3
6.1 6.2 6.4
7.8
5.6
6.3
158
Specialia
EXPERIENTIA 31/2
2.0 *
E280 mU/fraction
.
1.5-3
/'~
-10 pH 9
. ~.
8
*
7
x
/
1.0-2
....
**
t5 !
,,
0.5 1
e ~
_.
..
9
O0
]4
"
9
t
i
10
20
9
9
9
9
3'0
9
-o i
i
i~
40 Fraction No.
50
60
70
8'0
90
Fig. 2. Preparative free isoelectric focusing of E. coli extract (1.8 ml extract, containing 324 m U DFPase and 29 mg protein + 12 nfi 2% ampholine, pH 3-6, 41 h, 1000 V. • pH; ~, protein (E 1260J; cm~ 9 DFPase (mU/fraetion).
i n F i g u r e 4, t h e p H o p t i m u m is a t 8.3. T h e c o r r e s p o n d i n g k i n e t i c c o n s t a n t s for p a r a o x o n a s e i n E. coli t e s t e d w i t h p a r a o x o n a r e K m 5 • 10 -s M/1, tile p H o p t i m u m is a t 9.3. T h e e x t r a c t e d p h o s p h o r y l p h o s p h a t a s e s are rather u n s t a b l e , l o s i n g a b o u t 10 t o 30~ a c t i v i t y p e r d a y a t r o o m temperature. T h e i n s t a b i l i t y of t h e e n z y m e s a n d t h e l o w s u b s t r a t e a f f i n i t y m a k e E. coli D F P a s e a n d p a r a o x o n a s e u n s u i t a b l e tools for preparing efficient enzymatic protectors against o r g a n o p h o s p h a t e p o i s o n i n g of m a m m a l s .
1
1.5 "V mir~ vmole 1.0
0.5
,I
I00. . . .
2;0
' 6;0 400 DFP If/mole)
800 1/S
Fig. 3. LINEWEAVER-BoRK-p1ot of DFP-hydrolysis by E. coli DFPase. Automatic titrimetric assay in 10 ml 0.1 M NTaCl with 200 ~xl extract (10 mg protein), pH 8, 25~
Zusammen/assung. D i e E n t g i f t u n g v o n O r g a n o p h o s p h a t e n d u r c h E coli-Phosphophydrolasen w u r d e u n t e r s u c h t u n d e i n e D F P a s e (E.C. 3.8.2.1) u n d e i n e P a r a o x o n a s e (E.C. 3.1.1.2) n a c h g e w i e s e n . D i e T r e n n u n g d e r Phosphohydrolasen erfolgte durch Gelfiltration und die i s o e l e k t r i s c h e n P u n k t e w u r d e n in freier, p r / i p a r a t i v e r Fokussierung bestimmt. Ausserdem wurden Substrat- und pH-AbhSmgigkeit untersucht. R . ZECH a n d K . D. WIGAND
Physiologisch-chemisches Institut der Universitiit, Humboldtallee 7, D-34 GSttingen (German Federal Republic, BRD), 23 September 7974.
0.08 007 0.06
\
0.05 ~ K :=_
0.04 0.03
O.O2 0.01 r
0
8.5 ' 7.0 ' 7,5 d .0 8.5 ' ; 0 6
.5 I00 pH
Fig. 4. pH-dependenee of E. coli DFPase. Automatic titrimetrie method at 0.1 M substrate concentration in 0.1 M NaC1 with 200 Exl extract containing 10 mg protein.
1 E. D. ADRIAN, W. FELDBERG and B. A. KILBV, Br. J. Pharmae. 2, 56 (1947). 2 A. MAZnR, J. biol. Chem. 76d, 271 {1946). s K. B. AUGIJSTINSSO~, Biochim. biophys. Aeta 73, 303 (1954). R. Z E g and H. ENGELHARD,Biochem. Z. 343, 86 (1965). 5 R. ZEc~, Hoppe-Seyler's Z. physiol. Chem. 350, 1415 (1969). s R. ZECH and K. D. WIGAND,Z. kiln. Chem. klin. Bioehem. 71, 446 (1973). 7 R. ZECH and K. ZORCHER, Comp. Bioehem. Physiol. 48B, 427 (1974). s It. MelLwAIN, J. gen. Mierobiol. 2, 288 (1948). 9 T. WIELAND, P. DOSBERG and H. DETERMANN, Biochem. Z. 377, 303 (1963). :to V. MACKO and H. STEGEMANN, Analyt. Bioehem. 37, 186 (1970). 11 R. ZEC}I and K. Z*JRCHER, Life Sei. 13, 383 (1973).
Specialia
t h e n again, it m a y be t h a t t h e i n t r a n u c l e a r calcium t a k e s p a r t in i n t r a c e l l u l a r c a l c i u m kinetics. W h e t h e r t h e c h a n g e s are r e l a t e d t o m e c h a n i c a l muscle a c t i v i t y or are an e x p r e s s i o n of a d a p t a t i o n processes r e m a i n s unclear.
157
Rdsumd. L ' a n a l y s e p a r m i c r o s o n d e 61ectronique des cellules m u s c u l a i r e s m o n t r e de h a u t e s c o n c e n t r a t i o n s de c a l c i u m d a n s tes n o y a n x ; la c o n c e n t r a t i o n p e u t fitre modifi6e p h a r m a c o l o g i q u e m e n t ou p a r , p a c e m a k e r ~ 61ectrique R. YAROM, T. A. HALL a n d P. D. PETERS 15
15 We acknowledge the technical assistance of M. ScRiePs and S. SCHWARTZ.We thank B. GOLEKfor editorial help. This research was supported by the British Science Research Council, the Joint Research Fund of the Hebrew University-Hadassah Medical School, and an EMBO travelling fellowship for R.Y.
Department o/Pathology, Hebrew University-Hadassah Medical School, P.O. Box 7772, Jerusalem (Israel), and University o/ Cambridge, Cavendish Laboratory, Free School Lane, Cambridge CB2 3RQ ~England)s 27 May 7974.
Organophosphate-Detoxicating Enzymes in E. coli. Gelfiltration and Isoelectric Focusing of DFPase, Paraoxonase and Unspecific Phosphohydrolases Toxic o r g a n 0 p h o s p h a t e s , c o m m o n l y k n o w n as insecticides or as n e r v e gases, are i n h i b i t o r s of cholinesterases, thereby blocking nerve translnissionL Some hydrolytic e n z y m e s ( ' p h o s p h o r y l p h o s p h a t a s e s ' ) are c a p a b l e of s p l i t t i n g t h e a c i d a n h y d r i d e b o n d of o r g a n o p h o s p h a t e poisons, f o r m i n g n o n - t o x i c o r g a n o p h o s p h o r u s acid2, ~. The following p a p e r describes t h e e n z y m a t i c organop h o s p h a t e d e t o x i c a t i o n b y E. coli p h o s p h o h y d r o l a s e s . Two d e t o x i c a t i n g e n z y m e s were f m m d , a D F P a s e (E.C. 3.8.2.1) a n d a p a r a o x o n a s e (arylesterase, E.C. 3.1.1.2). D F P a s e - a c t i v i t y was d e t e r m i n e d b y 3 m e t h o d s w i t h d i - i s o p r o p y l f l u o r o p h o s p h a t e ( D F P ) as s u b s t r a t e : titrim e t r i c a l l y 4, m a n o m e t r i c a l l y 5 or b y m e a s u r i n g t h e liber-
Fig. 1. Geliiltration of E. coli extract on Sephadex G-200 (2.5 • 73 cm). 4 mi extract (2 g wet ceils) containing 73 mg protein and 562 mU DFPase were fraetionated at I~ flow rate: 0.2 ml/min. O, protein (E 1260J; emx 9 DFPase (mU/ml).
a t e d fluoride w i t h an i o n s e n s i t i v e electrode ~. P a r a o x o n a s e was t e s t e d colorimetrically w i t h 0 , 0 - d i e t h y l - 0 - p - n i t r o p h e n y l p h o s p h a t e (paraoxon)7. Alkaline a n d acid p h o s p h a t a s e were m e a s u r e d colorimetrically w i t h p - n i t r o p h e n y l p h o s p h a t e in 0.2 M Tris-buffer a t p H 9 a n d p i t 6, t h e a b s o r b a n c e a t 405 n m was r e a d a f t e r d i l u t i n g s a m p l e s with 1 M sodium hydroxide. Phosphodiesterase determin a t i o n s were p e r f o r m e d w i t h bis-p-nitrophenylphosphate a t p H 8 in t h e s a m e way. All t e s t s were p e r f o r m e d a t 25 ~ a c t i v i t y is e x p r e s s e d as 1 a m o l e s u b s t r a t e h y d r o l y z e d per min. E. coli K12sr e x t r a c t s were p r e p a r e d according to MCILwAIN 8. T h e e x t r a c t was f r a c t i o n a t e d b y gelfiltratioI1 on a S e p h a d e x G-200 columnl t h e elution d i a g r a m for p r o t e i n u n d D F P a s e is s h o w n in Figure 1. The d i s t r i b u t i o n coefficients (Kd-valus) of t h e 2 D F P a s e p e a k s were c a l c u l a t e d 9 a n d c o m p a r e d w i t h t h o s e of Other p h o s p h o h y d r o l a s e s in a second e x p e r i m e n t on t h e s a m e c o l u m n (Table I). D F P a s e a n d p a r a o x o n a s e could clearly be s e p e r a t e d b y gelfiltration; no o v e r l a p p i n g a c t i v i t y was d e t e c t e d : D F P a s e f r a c t i o n s did n o t h y d r o l y z e p a r a o x o n a n d vice versa. T h e r e was no h y d r o l y s i s of p - n i t r o p h e n y l p h o s p h a t e at acid or alkaline p i t or of bis-p-nitro-phenylphosphate b y e i t h e r t h e D F P a s e or p a r a o x o n a s e c o n t a i n i n g fractions. The p h o s p h o h y d r o l a s e s in E. coli e x t r a c t s were a d d i t i o n a l l y s u b j e c t e d to free p r e p a r a t i v e isoelectric f o c u s i n g l 0 , n . F o u r D F P a s e p e a k s w i t h is0electric p o i n t s of 5.3, 5.7, 6.1 a n d 7.8 were f o u n d (Figure 2). T h e isoelectric p o i n t s of t h e o t h e r p h o s p h o h y d r o l a s e s were det e r m i n e d in a n i d e n t i c a l w a y in a d i f f e r e n t e x p e r i m e n t , t h e isoelectric p o i n t s are listed in Table II. The p h o s p h o h y d r o l a s e s of E. coli s h o w e d similar or e v e n identical isoelectric points, so, in c o n t r a s t t o t h e gelfiltration e x p e r i m e n t , t h e y could n o t be s e p a r a t e d b y t h i s m e t h o d . Coli D F P a s e h a s a low s u b s t r a t e a f f i n i t y ( K m 1.7 • 10 -~ M/1 for D F P ) a n d shows p u r e MICHAELIS-MENTEN kinetic (Figure 3). T h e p H - d e p e n d e n c e of coli D F P a s e is s h o w n
Table I. Distribution coefficients (Kd-valus) of E. coli phosphohydrolases oI~ Sephadex G-200 (2.5 • 73 cm)
Table II. Isoelectric points of E. coli phosphohydrolases determined by free preparative isoelectrie focusing in 2% ampholine, pH 3-6
Enzyme
Kd-valus
Enzyme
DFPase Paraoxonase Acid phosphatase Alkaline phosphatase Phosphodiesterase
0.21 0.11 0.39 0.42 0.40
10 ~2
uJ
&
.E 0
ro
1
0
I00
150
200
250
300
350 ml
Eluate
0.31 0.55
0.66
DFPase Paraoxonase Acid phosphatase Alkaline phosphatase Phosphodiesterase
Isoelectric points 5.3 5.3 4.9 4.8 5.0
5.7 5.6 5.3 5.3 5.3
6.1 6.2 6.4
7.8
5.6
6.3
158
Specialia
EXPERIENTIA 31/2
2.0 *
E280 mU/fraction
.
1.5-3
/'~
-10 pH 9
. ~.
8
*
7
x
/
1.0-2
....
**
t5 !
,,
0.5 1
e ~
_.
..
9
O0
]4
"
9
t
i
10
20
9
9
9
9
3'0
9
-o i
i
i~
40 Fraction No.
50
60
70
8'0
90
Fig. 2. Preparative free isoelectric focusing of E. coli extract (1.8 ml extract, containing 324 m U DFPase and 29 mg protein + 12 nfi 2% ampholine, pH 3-6, 41 h, 1000 V. • pH; ~, protein (E 1260J; cm~ 9 DFPase (mU/fraetion).
i n F i g u r e 4, t h e p H o p t i m u m is a t 8.3. T h e c o r r e s p o n d i n g k i n e t i c c o n s t a n t s for p a r a o x o n a s e i n E. coli t e s t e d w i t h p a r a o x o n a r e K m 5 • 10 -s M/1, tile p H o p t i m u m is a t 9.3. T h e e x t r a c t e d p h o s p h o r y l p h o s p h a t a s e s are rather u n s t a b l e , l o s i n g a b o u t 10 t o 30~ a c t i v i t y p e r d a y a t r o o m temperature. T h e i n s t a b i l i t y of t h e e n z y m e s a n d t h e l o w s u b s t r a t e a f f i n i t y m a k e E. coli D F P a s e a n d p a r a o x o n a s e u n s u i t a b l e tools for preparing efficient enzymatic protectors against o r g a n o p h o s p h a t e p o i s o n i n g of m a m m a l s .
1
1.5 "V mir~ vmole 1.0
0.5
,I
I00. . . .
2;0
' 6;0 400 DFP If/mole)
800 1/S
Fig. 3. LINEWEAVER-BoRK-p1ot of DFP-hydrolysis by E. coli DFPase. Automatic titrimetric assay in 10 ml 0.1 M NTaCl with 200 ~xl extract (10 mg protein), pH 8, 25~
Zusammen/assung. D i e E n t g i f t u n g v o n O r g a n o p h o s p h a t e n d u r c h E coli-Phosphophydrolasen w u r d e u n t e r s u c h t u n d e i n e D F P a s e (E.C. 3.8.2.1) u n d e i n e P a r a o x o n a s e (E.C. 3.1.1.2) n a c h g e w i e s e n . D i e T r e n n u n g d e r Phosphohydrolasen erfolgte durch Gelfiltration und die i s o e l e k t r i s c h e n P u n k t e w u r d e n in freier, p r / i p a r a t i v e r Fokussierung bestimmt. Ausserdem wurden Substrat- und pH-AbhSmgigkeit untersucht. R . ZECH a n d K . D. WIGAND
Physiologisch-chemisches Institut der Universitiit, Humboldtallee 7, D-34 GSttingen (German Federal Republic, BRD), 23 September 7974.
0.08 007 0.06
\
0.05 ~ K :=_
0.04 0.03
O.O2 0.01 r
0
8.5 ' 7.0 ' 7,5 d .0 8.5 ' ; 0 6
.5 I00 pH
Fig. 4. pH-dependenee of E. coli DFPase. Automatic titrimetrie method at 0.1 M substrate concentration in 0.1 M NaC1 with 200 Exl extract containing 10 mg protein.
1 E. D. ADRIAN, W. FELDBERG and B. A. KILBV, Br. J. Pharmae. 2, 56 (1947). 2 A. MAZnR, J. biol. Chem. 76d, 271 {1946). s K. B. AUGIJSTINSSO~, Biochim. biophys. Aeta 73, 303 (1954). R. Z E g and H. ENGELHARD,Biochem. Z. 343, 86 (1965). 5 R. ZEc~, Hoppe-Seyler's Z. physiol. Chem. 350, 1415 (1969). s R. ZECH and K. D. WIGAND,Z. kiln. Chem. klin. Bioehem. 71, 446 (1973). 7 R. ZECH and K. ZORCHER, Comp. Bioehem. Physiol. 48B, 427 (1974). s It. MelLwAIN, J. gen. Mierobiol. 2, 288 (1948). 9 T. WIELAND, P. DOSBERG and H. DETERMANN, Biochem. Z. 377, 303 (1963). :to V. MACKO and H. STEGEMANN, Analyt. Bioehem. 37, 186 (1970). 11 R. ZEC}I and K. Z*JRCHER, Life Sei. 13, 383 (1973).
