150
Specialia
EXPERIENTIA 31/2
A F a c t o r w h i c h C o u n t e r a c t s the S t a b i l i z i n g A c t i v i t y of A c e t a t e ! o n o n O p h i o x i d a s e at A l k a l i n e p H W e h a v e reported t h a t L-amino acid oxidase (Lamino acid: O~ oxidoreductase, E C 1.4.3.2) from Crotalus adamanteus v e n o m exhibits a v a r i e t y of p H curves depending on the amino acids used as the substrate 1. This enzyme is highly unstable at high alkaline pH, t h o u g h acetate ion, as well as a large n u m b e r of aliphatic and a r o m a t i c m o n o c a r b o x y l i c acids, protects it from i n a c t i v a t i o n 2. I n this communication, evidence is presented t h a t beef liver contains a factor which p a r t i a l l y counteracts the effect of acetate ion. Materials and methods. Twice crystallized L-amino acid oxidase from Crotallus adamanteus v e n o m and the proteins used in compiling the d a t a in T a b l e I I were purchased from Sigma Chemical Co. t~reparation o/ a/actor. A p p r o x i m a t e l y 10 g of frozen beef liver was thawed, homogenized in 3 volumes of water, and passed t h r o u g h a double layer of cheese cloth. 20 ml of t h e h o m o g e n a t e was centrifuged at 39,000 g for 20 rain and 15.5 ml of the s u p e r n a t a n t was obtained. The s u p e r n a t a n t was t r e a t e d w i t h an equal v o l u m e of Cas(PO4) 2 gel (17.5 mg. solid/ml) and washed successively w i t h 30 ml of w a t e r and 15 ml of 0.02 M p h o s p h a t e buffer at p H 7.2. The gel was Muted w i t h 4 ml of 0.1 M p H 7.2 p h o s p h a t e buffer at 39,000 g for 10 min. 8 ml of the o b t a i n e d eluate was t r e a t e d w i t h an equal v o l u m e of
._.s o
s_
200 2.0
- t.9
1'0 ~2201Jg
~_>
Factor o
,7 1.8
o
IJA
i
0
2
4
i
6 Factor
o
,~~
i
8
s a t u r a t e d a m m o n i u m sulfate, and t h e preccipitate was collected after 10 min. The precipitate was dissolved in 8 ml of water, and 4 m l of h y d r o x y a p a t i t e (98.5 m g solid/ml) was added to the suspension. This was washed once w i t h 30 ml of water and finally Muted w i t h 4 ml of 0.1 iV/ p H 7.2 p h o s p h a t e buffer b y centrifugation at 39,000 g for 10 rain. The purification schedule and rec o v e r y of the factor is shown in Table I. Enzyme assay. I n t o the m a i n c o m p a r t m e n t of a double side a r m W a r b u r g flask 0.5 ml of 2.0 M sodium acetate (water solution has a p H 9.0), 1.0 ml of 0.1 M g l y c i n e - N a O H buffer, p H 9.0, and 0.5 ml of the factor suspended in t h e above buffer were placed. I n the case of control, the factor was replaced w i t h buffer. The 2 side arms contained 0.5 ml of 0.1 M L-arginine and 0.5 ml of crystalline e n z y m e solution containing 0.09 m g protein, respectively. All the reagents were prepared in the buffer except the L-amino acid oxidase, which was suspended in water. A 2 c m • 2 cm piece of filter paper, s a t u r a t e d w i t h 0.2 ml of 20% K O H , was placed in the center well to absorb carbon dioxide. The vessels were equilibrated at 37 ~ for 10 mili. The e n z y m e was t h e n t i p p e d into the main c o m p a r t m e n t of t h e flask and p r e i n c u b a t e d at p H 9.0 for 30 rain before the a d d i t i o n of substrate from the second side arm. The o x y g e n c o n s u m p t i o n in the first 12 rain was recorded and the e n z y m e a c t i v i t y expressed as F m o l e s of substrate o x i d i z e d / h / m g of e n z y m e protein. P r o t e i n c o n c e n t r a t i o n was d e t e r m i n e d by the m e t h o d of LOWRY et al.3. Results and discussion. As shown p r e v i o u s l y 1, 2, w h e n L-amino acid oxidase was p r e i n c u b a t e d for 30 min at p H 9.0 in the absence of substrate, t h e e n z y m e a c t i v i t y was abolished. However, the presence of 0.4 M sodium acetate during p r e i n c u b a t i o n c o m p l e t e l y p r o t e c t e d t h e e n z y m e from inactivation. As shown ill Figure 1, t h e a c t i v i t y of L-amino acid oxidase is c o m p l e t e l y p r o t e c t e d b y sodium acetate during p r e i n c u b a t i o n in the absence of the factor. Increasing concentrations of t h e factor decreases t h e p r o t e c t i v e a c t i v i t y of the acetate ion (Figure 1A). H o w e v e r , after c o u n t e r a c t i n g t h e p r o t e c t i v e a c t i v i t y of acetate a b o u t 40% (60% of the e n z y m e a c t i v i t y remains), t h e increasing c o n c e n t r a t i o n of the factor does n o t further influence t h e degree of inhibition. I t is unlikely t h a t t h e p h o s p h a t e ion, introduced w h e n eluting t h e factor from h y d r o x y a p a t i t e , counteracts t h e acetate since t h e final
1() //l 20IJg
Fig. 1. Effect of concentration of the factor on the p r o t e c t i v e action of acetate of L-amino acid oxidase. D u r i n g p r e i n c u b a t i o n of L-amino acid oxidase in p H 9.0 for 30 min, 0.4 M a c e t a t e was present; Detailed conditions are described under Methods.
1 W. K. PAIK and S. KIM, Biochim. biophys. Acta !76, 66 (1965). W. I~. PAIK and S. KIM, Bioehim. biophys. A c t a 739, 49 (1967}. a O. H. LOWRY, N. J. ROSEBROUGH, A. L. FARR and R. J. RANDALL, J. biol. Chem. 793, 265 (1951).
Table I. Purification of the factor from beef l i v e r a Purification step
Whole h o m o g e n a t e S u p e r n a t a n t a t 39,000 g for 20 m i n E l u a t e from c a l c i u m p h o s p h a t e gel P r e c i p i t a t e from a m m o n i u m sulfate E l u a t e from h y d r o x y a p a t i t e
Volume
Protein
Factor activity
(ml)
(mg/ml)
(units) b
20.0 15.5 8.0 8.0 4.9
58.9 38.8 8.6 4.4 2.1
41.0 73.5 138.9 312.5 500.0
T o t a l units
Yield
Purification
(%) 48,298 44,203 9,556 11,000 5,145
100.0 91.5 19.8 22.8 10.7
1.0 1.8 3.4 7.6 12.2
20 days frozen beef liver w a s used. b Unit activity is defined as m g of the factor to decrease the effect of acetate ion from 1 0 0 % For detail see the text.
to 8 0 % .
15.2. 1975
151
SPecialia
"Fable II. Specificity of the factor to counteract the effect of acetate on inactivation of L-amino acid oxidase at pH 9.0 Compound
Amount (mg)
None Factor (isolated from beef liver) Acetone powder extract of Bact. cadavefis Extract of acetone powder of pigeon liver Lysine-rieh histone Globulin Protamine Arginine-rich histone Lysozyme Trypsin inhibitor Polylysine Ribonuclease Bovine serum albumin
0.01 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.i0 0.10
Remaining enzyme activity (Fmole/h/mg enzyme protein)
%
226.8 150.7 153.5 155.3 232.8 209.9 222.1 216.8 218.0 215.2 219.1
100.0 66.4 67.7 67.8 102.6 92.5 97.9 95.6 96.1 94.9 96.7 97.6 98.0
221.4 222.2
The conditions are described in the text.
c o n c e n t r a t i o n of p h o s p h a t e w a s less t h a n 10 -4 M . I n a p r e v i o u s s t u d y , p h o s p h a t e in t h i s c o n c e n t r a t i o n h a d no effect ~. W h e n t h e v a l u e s in Figure 1A were p l o t t e d as l o g a r i t h m of p e r c e n t a c t i v i t y vs. t h e a m o u n t of t h e f a c t o r (Figure 113), a s t r a i g h t line was o b t a i n e d a t regions w h e r e t h e c o n c e n t r a t i o n of t h e f a c t o r h a d a n effect on t h e degree of c o u n t e r a c t i o n . Therefore, as in t h e case of T a b l e I, u n i t a c t i v i t y of t h e f a c t o r w a s d e f i n e d as m g of t h e f a c t o r n e c e s s a r y to decrease t h e l o g a r i t h m i c v a l u e f r o m 2 to 1.9. This c o r r e s p o n d s to a decrease of e n z y m e a c t i v i t y f r o m 100% t o 80%. A reciprocal p l o t of a c e t a t e c o n c e n t r a t i o n a n d t h e r a t e of L-amino acid o x i d a s e is s h o w n in F i g u r e 2. I t is e v i d e n t f r o m t h i s figure t h a t t h e f a c t o r a n d a c e t a t e a c t c o m p e t i tively. H o w e v e r , since t h e c o u n t e r a c t i o n does n o t increase i n d e f i n i t e l y w i t h increase of t h e f a c t o r c o n c e n t r a t i o n (Figure 1), t h i s i n h i b i t i o n is of a p a r t i a l l y c o m p e t i t i v e n a t u r e 4. Several lines of e v i d e n c e suggest t h a t t h e f a c t o r is a protei~ n a t u r e : a) it is i n a c t i v a t e d b y h e a t i n g a t 60 ~ for 5 rain; b) t h e f a c t o r was p u r i f i e d b y c o n v e n t i o n a l m e t h o d s of e n z y m e p u r i f i c a t i o n . Various p r o t e i n s were t e s t e d for
1
t h e i r c a p a c i t y t o c o u n t e r a c t t h e p r o t e c t i v e effect of a c e t a t e on L-amino acid oxidase. As s h o w n ill Table II, histone, globulin, p r o t a m i n e , lysozyme, t r y p s i n inhibitor. polylysine, a l b u m i n a n d ribonuclease h a v e no effect. There seems to be no relation between basicity of protein and the inhibitory activity, since albumin is acidic and the rest of the above proteins are basic. O n the other hand, acetone powder extracts of Bact. cadaveris and of pigeon liver demonstrate counteracting ability. Therefore, it appears that the factor is quite c o m m o n in nature. W h e n the factor was preincubated with acetate-14C and this mixture was passed through Bio-Gel P-10, it was found that all of the acetate-14C was recovered in the same position as if it alone were present during the preincubation, as was the protein peak which contained no radioactivity. The factor itself was excluded by this gel, indicating molecular weight greater than I0,000. These results suggest t h a t t h e f a c t o r n e i t h e r d e s t r o y s n o r c o m b i n e s w i t h t h e acetate-14C. I t is h i g h l y p r o b a b l e t h a t t h e f a c t o r m i g h t r e g u l a t e t h e effect of a c e t a t e on t h e L-amino acid o x i d a s e in s o m e allosteric fashion. F u r t h e r i n v e s t i g a t i o n into t h i s p r o b l e m will b e c o n t i n u e d . P r e s e n t l y , t h e biological significance of t h e s e f i n d i n g s r e m a i n clouded, however, t h e a p p a r e n t c o m m o n o c c u r r e n c e of t h i s f a c t o r in n a t u r e m i g h t i n d i c a t e a f o r m of L-amino acid o x i d a s e r e g u l a t o r y m e c h a n i s m b a s e d on t h e p r o p o r t i o n of f a c t o r : a c e t a t e ion in t h e liver of h i g h e r animals.
V Rdsumd. L a L-aminoacide o x y d a s e de v e n i n de s e r p e n t est tr6s i n s t a b l e en milieu alcalin et les ions d ' a c e t a t e s o u t i e n n e n t c e t t e i n a c t i v a t i o n . Le foie de b0euf c o n t i e n t un f a c t e u r qui i n h i b e p a r t i e l l e m e n t l ' e f f e t des ions d ' a c e t a t e . C e t t e i n h i b i t i o n est en p a r t i e de t y p e comp6titif.
0.02
0.01
W.
0
10 1
20
30
Fig. 2. Reciprocal plot of acetate concentration and the rat of Lamino acid oxidase. 10 tzg of the factor were used, and Vmax value in the figure was calculated as 285.7 ~xmoleL-arginine oxidized/h/mg enzyme protein.
K. PAIK
a n d S. KIM
The Fels Research Institute and Department of Biochemistry, Temple University School o/ Medicine, Philadelphia (Pennsylvania 19140, USA), 25 August 1974.
4 M. Dlxot~ and E. C. WEBB, Enzymes (Longmans, Green and Co. Lts., London 1962), p. 320.
Specialia
EXPERIENTIA 31/2
A F a c t o r w h i c h C o u n t e r a c t s the S t a b i l i z i n g A c t i v i t y of A c e t a t e ! o n o n O p h i o x i d a s e at A l k a l i n e p H W e h a v e reported t h a t L-amino acid oxidase (Lamino acid: O~ oxidoreductase, E C 1.4.3.2) from Crotalus adamanteus v e n o m exhibits a v a r i e t y of p H curves depending on the amino acids used as the substrate 1. This enzyme is highly unstable at high alkaline pH, t h o u g h acetate ion, as well as a large n u m b e r of aliphatic and a r o m a t i c m o n o c a r b o x y l i c acids, protects it from i n a c t i v a t i o n 2. I n this communication, evidence is presented t h a t beef liver contains a factor which p a r t i a l l y counteracts the effect of acetate ion. Materials and methods. Twice crystallized L-amino acid oxidase from Crotallus adamanteus v e n o m and the proteins used in compiling the d a t a in T a b l e I I were purchased from Sigma Chemical Co. t~reparation o/ a/actor. A p p r o x i m a t e l y 10 g of frozen beef liver was thawed, homogenized in 3 volumes of water, and passed t h r o u g h a double layer of cheese cloth. 20 ml of t h e h o m o g e n a t e was centrifuged at 39,000 g for 20 rain and 15.5 ml of the s u p e r n a t a n t was obtained. The s u p e r n a t a n t was t r e a t e d w i t h an equal v o l u m e of Cas(PO4) 2 gel (17.5 mg. solid/ml) and washed successively w i t h 30 ml of w a t e r and 15 ml of 0.02 M p h o s p h a t e buffer at p H 7.2. The gel was Muted w i t h 4 ml of 0.1 M p H 7.2 p h o s p h a t e buffer at 39,000 g for 10 min. 8 ml of the o b t a i n e d eluate was t r e a t e d w i t h an equal v o l u m e of
._.s o
s_
200 2.0
- t.9
1'0 ~2201Jg
~_>
Factor o
,7 1.8
o
IJA
i
0
2
4
i
6 Factor
o
,~~
i
8
s a t u r a t e d a m m o n i u m sulfate, and t h e preccipitate was collected after 10 min. The precipitate was dissolved in 8 ml of water, and 4 m l of h y d r o x y a p a t i t e (98.5 m g solid/ml) was added to the suspension. This was washed once w i t h 30 ml of water and finally Muted w i t h 4 ml of 0.1 iV/ p H 7.2 p h o s p h a t e buffer b y centrifugation at 39,000 g for 10 rain. The purification schedule and rec o v e r y of the factor is shown in Table I. Enzyme assay. I n t o the m a i n c o m p a r t m e n t of a double side a r m W a r b u r g flask 0.5 ml of 2.0 M sodium acetate (water solution has a p H 9.0), 1.0 ml of 0.1 M g l y c i n e - N a O H buffer, p H 9.0, and 0.5 ml of the factor suspended in t h e above buffer were placed. I n the case of control, the factor was replaced w i t h buffer. The 2 side arms contained 0.5 ml of 0.1 M L-arginine and 0.5 ml of crystalline e n z y m e solution containing 0.09 m g protein, respectively. All the reagents were prepared in the buffer except the L-amino acid oxidase, which was suspended in water. A 2 c m • 2 cm piece of filter paper, s a t u r a t e d w i t h 0.2 ml of 20% K O H , was placed in the center well to absorb carbon dioxide. The vessels were equilibrated at 37 ~ for 10 mili. The e n z y m e was t h e n t i p p e d into the main c o m p a r t m e n t of t h e flask and p r e i n c u b a t e d at p H 9.0 for 30 rain before the a d d i t i o n of substrate from the second side arm. The o x y g e n c o n s u m p t i o n in the first 12 rain was recorded and the e n z y m e a c t i v i t y expressed as F m o l e s of substrate o x i d i z e d / h / m g of e n z y m e protein. P r o t e i n c o n c e n t r a t i o n was d e t e r m i n e d by the m e t h o d of LOWRY et al.3. Results and discussion. As shown p r e v i o u s l y 1, 2, w h e n L-amino acid oxidase was p r e i n c u b a t e d for 30 min at p H 9.0 in the absence of substrate, t h e e n z y m e a c t i v i t y was abolished. However, the presence of 0.4 M sodium acetate during p r e i n c u b a t i o n c o m p l e t e l y p r o t e c t e d t h e e n z y m e from inactivation. As shown ill Figure 1, t h e a c t i v i t y of L-amino acid oxidase is c o m p l e t e l y p r o t e c t e d b y sodium acetate during p r e i n c u b a t i o n in the absence of the factor. Increasing concentrations of t h e factor decreases t h e p r o t e c t i v e a c t i v i t y of the acetate ion (Figure 1A). H o w e v e r , after c o u n t e r a c t i n g t h e p r o t e c t i v e a c t i v i t y of acetate a b o u t 40% (60% of the e n z y m e a c t i v i t y remains), t h e increasing c o n c e n t r a t i o n of the factor does n o t further influence t h e degree of inhibition. I t is unlikely t h a t t h e p h o s p h a t e ion, introduced w h e n eluting t h e factor from h y d r o x y a p a t i t e , counteracts t h e acetate since t h e final
1() //l 20IJg
Fig. 1. Effect of concentration of the factor on the p r o t e c t i v e action of acetate of L-amino acid oxidase. D u r i n g p r e i n c u b a t i o n of L-amino acid oxidase in p H 9.0 for 30 min, 0.4 M a c e t a t e was present; Detailed conditions are described under Methods.
1 W. K. PAIK and S. KIM, Biochim. biophys. Acta !76, 66 (1965). W. I~. PAIK and S. KIM, Bioehim. biophys. A c t a 739, 49 (1967}. a O. H. LOWRY, N. J. ROSEBROUGH, A. L. FARR and R. J. RANDALL, J. biol. Chem. 793, 265 (1951).
Table I. Purification of the factor from beef l i v e r a Purification step
Whole h o m o g e n a t e S u p e r n a t a n t a t 39,000 g for 20 m i n E l u a t e from c a l c i u m p h o s p h a t e gel P r e c i p i t a t e from a m m o n i u m sulfate E l u a t e from h y d r o x y a p a t i t e
Volume
Protein
Factor activity
(ml)
(mg/ml)
(units) b
20.0 15.5 8.0 8.0 4.9
58.9 38.8 8.6 4.4 2.1
41.0 73.5 138.9 312.5 500.0
T o t a l units
Yield
Purification
(%) 48,298 44,203 9,556 11,000 5,145
100.0 91.5 19.8 22.8 10.7
1.0 1.8 3.4 7.6 12.2
20 days frozen beef liver w a s used. b Unit activity is defined as m g of the factor to decrease the effect of acetate ion from 1 0 0 % For detail see the text.
to 8 0 % .
15.2. 1975
151
SPecialia
"Fable II. Specificity of the factor to counteract the effect of acetate on inactivation of L-amino acid oxidase at pH 9.0 Compound
Amount (mg)
None Factor (isolated from beef liver) Acetone powder extract of Bact. cadavefis Extract of acetone powder of pigeon liver Lysine-rieh histone Globulin Protamine Arginine-rich histone Lysozyme Trypsin inhibitor Polylysine Ribonuclease Bovine serum albumin
0.01 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.i0 0.10
Remaining enzyme activity (Fmole/h/mg enzyme protein)
%
226.8 150.7 153.5 155.3 232.8 209.9 222.1 216.8 218.0 215.2 219.1
100.0 66.4 67.7 67.8 102.6 92.5 97.9 95.6 96.1 94.9 96.7 97.6 98.0
221.4 222.2
The conditions are described in the text.
c o n c e n t r a t i o n of p h o s p h a t e w a s less t h a n 10 -4 M . I n a p r e v i o u s s t u d y , p h o s p h a t e in t h i s c o n c e n t r a t i o n h a d no effect ~. W h e n t h e v a l u e s in Figure 1A were p l o t t e d as l o g a r i t h m of p e r c e n t a c t i v i t y vs. t h e a m o u n t of t h e f a c t o r (Figure 113), a s t r a i g h t line was o b t a i n e d a t regions w h e r e t h e c o n c e n t r a t i o n of t h e f a c t o r h a d a n effect on t h e degree of c o u n t e r a c t i o n . Therefore, as in t h e case of T a b l e I, u n i t a c t i v i t y of t h e f a c t o r w a s d e f i n e d as m g of t h e f a c t o r n e c e s s a r y to decrease t h e l o g a r i t h m i c v a l u e f r o m 2 to 1.9. This c o r r e s p o n d s to a decrease of e n z y m e a c t i v i t y f r o m 100% t o 80%. A reciprocal p l o t of a c e t a t e c o n c e n t r a t i o n a n d t h e r a t e of L-amino acid o x i d a s e is s h o w n in F i g u r e 2. I t is e v i d e n t f r o m t h i s figure t h a t t h e f a c t o r a n d a c e t a t e a c t c o m p e t i tively. H o w e v e r , since t h e c o u n t e r a c t i o n does n o t increase i n d e f i n i t e l y w i t h increase of t h e f a c t o r c o n c e n t r a t i o n (Figure 1), t h i s i n h i b i t i o n is of a p a r t i a l l y c o m p e t i t i v e n a t u r e 4. Several lines of e v i d e n c e suggest t h a t t h e f a c t o r is a protei~ n a t u r e : a) it is i n a c t i v a t e d b y h e a t i n g a t 60 ~ for 5 rain; b) t h e f a c t o r was p u r i f i e d b y c o n v e n t i o n a l m e t h o d s of e n z y m e p u r i f i c a t i o n . Various p r o t e i n s were t e s t e d for
1
t h e i r c a p a c i t y t o c o u n t e r a c t t h e p r o t e c t i v e effect of a c e t a t e on L-amino acid oxidase. As s h o w n ill Table II, histone, globulin, p r o t a m i n e , lysozyme, t r y p s i n inhibitor. polylysine, a l b u m i n a n d ribonuclease h a v e no effect. There seems to be no relation between basicity of protein and the inhibitory activity, since albumin is acidic and the rest of the above proteins are basic. O n the other hand, acetone powder extracts of Bact. cadaveris and of pigeon liver demonstrate counteracting ability. Therefore, it appears that the factor is quite c o m m o n in nature. W h e n the factor was preincubated with acetate-14C and this mixture was passed through Bio-Gel P-10, it was found that all of the acetate-14C was recovered in the same position as if it alone were present during the preincubation, as was the protein peak which contained no radioactivity. The factor itself was excluded by this gel, indicating molecular weight greater than I0,000. These results suggest t h a t t h e f a c t o r n e i t h e r d e s t r o y s n o r c o m b i n e s w i t h t h e acetate-14C. I t is h i g h l y p r o b a b l e t h a t t h e f a c t o r m i g h t r e g u l a t e t h e effect of a c e t a t e on t h e L-amino acid o x i d a s e in s o m e allosteric fashion. F u r t h e r i n v e s t i g a t i o n into t h i s p r o b l e m will b e c o n t i n u e d . P r e s e n t l y , t h e biological significance of t h e s e f i n d i n g s r e m a i n clouded, however, t h e a p p a r e n t c o m m o n o c c u r r e n c e of t h i s f a c t o r in n a t u r e m i g h t i n d i c a t e a f o r m of L-amino acid o x i d a s e r e g u l a t o r y m e c h a n i s m b a s e d on t h e p r o p o r t i o n of f a c t o r : a c e t a t e ion in t h e liver of h i g h e r animals.
V Rdsumd. L a L-aminoacide o x y d a s e de v e n i n de s e r p e n t est tr6s i n s t a b l e en milieu alcalin et les ions d ' a c e t a t e s o u t i e n n e n t c e t t e i n a c t i v a t i o n . Le foie de b0euf c o n t i e n t un f a c t e u r qui i n h i b e p a r t i e l l e m e n t l ' e f f e t des ions d ' a c e t a t e . C e t t e i n h i b i t i o n est en p a r t i e de t y p e comp6titif.
0.02
0.01
W.
0
10 1
20
30
Fig. 2. Reciprocal plot of acetate concentration and the rat of Lamino acid oxidase. 10 tzg of the factor were used, and Vmax value in the figure was calculated as 285.7 ~xmoleL-arginine oxidized/h/mg enzyme protein.
K. PAIK
a n d S. KIM
The Fels Research Institute and Department of Biochemistry, Temple University School o/ Medicine, Philadelphia (Pennsylvania 19140, USA), 25 August 1974.
4 M. Dlxot~ and E. C. WEBB, Enzymes (Longmans, Green and Co. Lts., London 1962), p. 320.
