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Erythrocyte acetylcholinesterase in duchenne muscular dystrophy
241
Clinica Chimica Acta, 105 (1980) 241-247 0 Elsevier/North-Holland Biomedical Press
CCA 1428
ERYTHROCYTE DYSTROPHY
ACETYLCHOLINESTERASE
HIDEKI IGISU a-*, SHIRO SANJI MIYOSHINO d
MAWATARI
IN DUCHENNE MUSCULAR
b, YOSHIGORO
KUROIWA
c and
a Institute
ofHealthScienceand DepartmentofNeurology, Kyushu University, Fukuoka 812 (Japan), b Health Administration Center, Kyushu Institute of Technology, and Department of Neurology, Kyushu University, Fukuoka (Japan), c Department of Neurology, Faculty of Medicine, Kyushu University, Fukuoka 812 (Japan) and d Department of Pediatrics, Nishi-Beppu Hospital, Beppu (Japan)
(Received
November
26th,
1979)
Summary The activity and the activation energy (25-43°C) of acetylcholinesterase did not differ between erythrocytes from patients with Duchenne muscular dystrophy (DMD) and controls. The Hill coefficient for the inhibition of the enzyme activity by fluoride was 1 in both groups. The dose of fluoride for 50% inhibition of the enzyme activity differed between samples prepared in different ways, but not between DMD and controls. The present results suggest that there is no gross abnormality in erythrocyte acetylcholinesterase or its environment in the erythrocyte membrane in DMD.
Introduction Although often conflicting, various abnormalities of erythrocytes have been described in Duchenne muscular dystrophy (DMD). Erythrocyte membranes, especially in man, have a high activity of acetylcholinesterase (AchE), an enzyme localized on the outer side of the membrane [l]. The physiological role of this enzyme in erythrocytes has not been defined but it seems a useful probe for the study of membranes [ 2,3]. To our knowledge, there has been one study on erythrocyte AchE in DMD which described abnormal response to inhibitors [ 41. High activity of muscle AchE was reported in DMD [5]. We therefore studied the characteristics of erythrocyte AchE in DMD.
* Correspondence cine,
Kyushu
should University.
be addressed Fukuoka
to: 812,
Hid&i Japan.
Igisu,
M.D.,
Department
of Neurology.
Faculty
of Medi-
242
Subjects and methods
All subjects were males. Experiments 1 and 2 were done on separate days, but within each experiment samples from patients with DMD and age-matched controls (patients with cerebral palsy in experiment 1 and patients with asthma in experiment 2) were obtained on the same day at almost the same time (just before lunch) and processed simultaneously. In experiment 1, all subjects were in the same institution. For anticoagulation, EDTA-2Na (experiment 1) or heparin-Na (experiment 2) was used. The blood samples were immediately placed on ice until 5 h (experiment 1) or 2 h (experiment 2) later, respectively, when the preparation of the samples was started. In experiment 1, we made ghosts with a method similar to Brown et al. [6] : after plasma and buffy coat were removed, the erythrocytes were washed three times with isotonic saline. The erythrocytes were hemolyzed with 2 mmol/l Tris-HCl, pH 7.4, containing 0.02 mmol/l EDTA-2Na. The ghosts were sedimented at 20 000 X g for 20 min and washed 5 times with the same buffer. After the second and the last centrifugation, the ghosts were homogenized with a Teflon pestle. During this procedure the temperature was kept at 4°C. The ghosts, suspended in the buffer (the protein concentration approximately 5 mg/ml), were kept at -80°C until use when the protein concentration was made 2 mg/ml with 2 mmol/l Tris-HCl, pH 7.4. Then equal volumes of 0.1 mol/l phosphate buffer, pH 7.4, or the same buffer containing 2% Triton X-100 were added, making the final protein concentration of the ghost suspension 1 mg/ml, and kept on ice overnight. In experiment 2, the erythrocytes were washed twice with isotonic saline and then once with 0.1 mol/l phosphate buffer, pH 7.4. The erythrocytes were resuspended in the same buffer to make the hemoglobin concentration 4.0 g/dl, kept on ice overnight and hemolyzed with freeze and thaw in dry ice-acetone. The AchE activity was assayed by a slight modification of the method of Ellman et al. [ 71. The reaction mixture consisted of 3 ml of 0.1 mol/l phosphate buffer, pH 7.4, containing 0.25 mmol/l 5,5’dithiobis-2-nitrobenzoic acid, 50 ,YI of 40 mmol/l acetylthiocholine iodide, and 20 ~1 of ghost suspension or 10 ~1 of hemolysate. An appropriate volume (5-35 ~1) of 240 mmol/l NaF was added when necessary. Addition of 35 ~1 of H,O or 240 mmol/l NaCl caused no change in the enzyme activity. The change in absorbance at 412 nm was observed with a spectrophotometer (Hitachi 200-20) equipped with a recorder (Beckman-Toshiba U-125M). The temperature of the buffer and the cuvette holder was controlled with a circulation bath (Lauda KSRD). The protein concentration of the ghost suspension was measured by the method of Lowry et al. [8] and the hemoglobin concentration of the erythrocyte suspension by the cyanmethemoglobin method [9]. The activation energy and the Hill coefficient for the inhibition of the enzyme activity by fluoride were obtained graphically according to the following equations: lnK=-EIRT+a,
where K is the reaction rate; R is gas constant; constant; E is the activation energy.
T is absolute
temperature;
a is a
243 LN ACE~YL~HOLINESTERASE
ACTIVITY
244
TABLE
I
AchE
ACTIVITY
(25*C)
AND
ACTIVATION
ENERGY
(25-43°C)
i. SD.1
Controls
DMD
Kxpcrimen
(MEAN
t 1 (ghosts)
N
fi
5
age
c S.D.)
(mean
activity
(pmoles
Triton
14.0 thiocholine
X-100
activation
energy
hydrolized/mg
(~imoles
i 5.7
proteinlmin)
(-_)
1.96
+ 0.18
2.08
(+I
1.94
+ 0.19
2.12
+ 0.17
2.40
+ 0.27
2.22
? 0.11
(caI/molf
Experimer2 t 2 (tz~m~t~,sates~ N age (mean + S.D.) activity
12.7
-? 5.1
6
5 9.4
thiocholine
k 0.17
t
1.9
hydrolizedfg 30.2
10.8
f 0.3
30.5
+ 1.6
Hbimin)
+ 2.5
log V/(V, - V) = log &.s - n log(l) where V, is the reaction velocity without fluoride; V is the reaction velocity with fluoride; K0,5 is the fluoride concentration when V/( V,-- V) is equal to 1.0, i.e., the dose of fluoride for 50% inhibition of the enzyme activity; I is the concentration of fluoride; n is the Frill coefficient. Results The AchE activity at 25°C showed no significant difference between DMD and controls either in hemolysates or ghosts with Triton X-100, or without (Table I). The activation energy (25-43’C) of the ghost AchE obtained from the Arrhenius plot (Fig. 1) did not differ between DMD and controls (Table I). When the AchE activity (at 25°C) was plotted against the dose of NaF, a
TABLE HILL
II COEFFICIENT
FOR
THE
INHIBITION
OF
AchE
BY
FLUORIDE
AND
Kg
5 AT
S.D.) -. __..._
--
DMD -...
.__
_
Experiment Hill
I (ghosts)
coefficient Triton
k’O.5
X-100
(-_)
1.01
-1 0.04
1.00
r 0.03
(+)
1.00
-t 0.04
0.99
f 0.04
(-_)
0.97
* 0.03
0.96
(+)
0.91
f 0.02
0.91
i 0.03
0.97
t 0.04 --
0.64
(mmoI/l)
Triton
X-100
Experiment 2 (hemolysates) Hill coefficient k’o.5 Number KO.5
0.67
and age of the subjects
is the
* Different Different
dose
of fluoride
@
< 0.01)
@
< 0.001)
*
0.96
(mmoI/lf
--.--
**
-~.-_
Controls
are the same
for
from from
**
50%
the the
inhibition
Ko.5 h’O.5
without
f 0.02 *
f 0.02
i 0.04 **
+ 0.03 .-.
as in Table of
AchE
Triton
of ghosts
with
I. activity. X-100. or without
Triton
X-100.
_
25OC
(MEAN
f
12
n
1
C-I
2
3
2
3
\
1
2
2
\
2
1
3 1
l
2
l2
2
1
c-3 kL?
.5
1
1
.5
n
3
1
c-2
2
~
1
1 .5 IIY!z?
2 D-3
1
2
1
2
2
Fig, 2. Effect of N@ on ghost A&E activity. D 1-5 ad activity @moles thiocboline hydrolyzed/mg protein/min).
1
2
1 .5
2
1
‘=
\
D-2
1 .5 L.YIL 1
2
1
2
123
1
2
3
rl
\
L
\-,
1 -
2
I 1
1
2
1
1
3 mM NaF
3
2
C-5
2
h
1
1
2
3 mM NaF
C l-6 are fame as in Fig. 1. The abscissa indicates concentration of NaF (mnolfl) Inset: the same data were replotted acCWding to the Hill WUatiOD.
3
2
3
1
2
and the ordinate
AchE
246
hyperbolic curve was obtained and the controls (Fig. 2). A significant difference of Ko,s was and between ghosts with and without ence between DMD and controls (Table
Hill coefficient
was 1 in both DMD and
noted between hemolysates and ghosts Triton X-100, but there was no differII).
Discussion It was reported that the extent of suppression of the AchE activity by a single dose of inhibitors including fluoride was abnormal in erythrocytes in DMD [4]. On the other hand, the Hill coefficient for the inhibition by fluoride of erythrocyte AchE activity appears a very sensitive indicator of the membrane characteristics [lO,ll]. We therefore studied the effect of fluoride on AchE activity with various doses of the drug and did Hill plot analysis. The dose-response curve was not sigmoid and the Hill coefficient was 1 in DMD and control erythrocytes, indicating no allosteric inhibition in either group. The dose of fluoride for a 50% inhibition of AchE activity differed between hemolysates and ghosts, or between ghosts with Triton X-100 and without it, showing that difference in preparation of samples affects the extent of suppression of the enzyme activity. But no difference was found between DMD and controls. In several previous studies on erythrocytes in DMD, samples from adults were included in the controls. However, erythrocytes from children and adults differ in various aspects [ 12-141. Differences in procedures for preparing ghosts has profound effects on membrane characteristics [ 15,161, and this may be the cause of the discrepancies in the findings in erythrocytes in DMD [14, 161. We, therefore, (1) used sex- and age-matched controls, (2) obtained samples from patients and controls at almost the same time on the same day, (3) processed the samples and assayed the enzyme activity from the two groups simultaneously, and (4) examined hemolysates as well as ghosts. The activation energy of an enzyme-catalysed reaction is considered an indicator of the conformation of the active site and it may be affected by the membrane fluidity [ 171. The Hill coefficient for the inhibition of AchE by fluoride is also affected by the membrane characteristics [ 10,111. We did not see any abnormality in these parameters or in the AchE activity in erythrocytes in DMD within the temperature range we studied. Thus, there seems to be no gross abnormality in the AchE or its environment in the erythrocyte membrane in DMD. Acknowledgements We thank Dr. Hamasaki (Department of Biochemistry, School of Medicine, Fukuoka University) for valuable comments, and Dr. Nishima (MinamiFukuoka Hospital) for kind cooperation. This work was supported by grants from the Ministry of Health and Welfare, Japan.
247
References Niday,
E., Wang,
Aloni,
B. and
Jarrett,
H.W.
Goedde.
and
H.W..
(1977)
Klin.
5
Kar,
6
Brown,
7
Ellman,
C.S.
Livne,
N.C.
and
Alaupovic,
(1974)
55,
J.T.
(1976)
H.G.,
Das,
Bioehim.
Biophys,
Biophys.
Acta
Biochim.
Biophys.
P.K.,
Agarwal.
339,
Acta
469,
180-193
359-366 Acta
D.P..
448,
Lang,
314-324
H..
Wiirzburg.
U. and
Beckmann,
R.
Pharmacol.
7,
215-217
Pearson,
C.M.
(1973)
Chattopadhyay,
G.L.,
P. (1977)
Biochim.
Penniston.
Benkmann,
Wschr.
H.D.,
and A.
Courtney,
Biochem.
S.K. K.D.,
and
Med.
Pat&,
Anders.
V.
A.B. and
7. 452459 (1967)
Science
Featherstone,
157.1577-1578
R.M.
(1961)
Biochem.
88-95 8
Lowry,
9
Dacie,
O.H., J.V.
Rosebrough, and
Lewis,
N.J., S.M.
Fan,
(1968)
A.L.
and
Randall,
in Practical
R.J.
(1951)
Haematology.
J. Biol.
4th
Ed.,
pp.
Chem. 37-39,
193,
265-275
J. & A.
Churchill,
London 10
Farias,
R.N.,
Bloj,
B.,
Morero,
Farias,
R.N.
R.D.,
Sineriz,
F. and
Trucco,
R.E.
(1975)
Biochitn.
231-251 11
Mendosa.
12
Kobayashi.
13
I&u,
H..
Mawatari.
14
Igisu.
H..
Mawatari,
15
Hanahan,
16
Roses,
17
McMurchie,
D. and T.,
D.J.
A.D.
Mawatari,
and
E.J.
and
J. Biol.
Kuroiwa,
Kuroiwa,
Y.
(1979)
S. and
Kuroiwa,
Y.
(1979)
CIin.
J.E.
Chim.
R&on,
(1978) Acta
J.K.
Chem.
Y.
S. and Ekholm,
(1979)
(1978)
S. and
Arch. 95,
(1979)
(1978)
253.62494254 Clin.
Chim.
Neurology Ann.
Neural.
Biochem.
Acta
85.
259-266
29,992-995 6, 86
Biophys.
187,170-179
69.-73 Biochim.
Biophys.
Acta
554,
364..-374
Biophys.
Acta
415,
Clinica Chimica Acta, 105 (1980) 241-247 0 Elsevier/North-Holland Biomedical Press
CCA 1428
ERYTHROCYTE DYSTROPHY
ACETYLCHOLINESTERASE
HIDEKI IGISU a-*, SHIRO SANJI MIYOSHINO d
MAWATARI
IN DUCHENNE MUSCULAR
b, YOSHIGORO
KUROIWA
c and
a Institute
ofHealthScienceand DepartmentofNeurology, Kyushu University, Fukuoka 812 (Japan), b Health Administration Center, Kyushu Institute of Technology, and Department of Neurology, Kyushu University, Fukuoka (Japan), c Department of Neurology, Faculty of Medicine, Kyushu University, Fukuoka 812 (Japan) and d Department of Pediatrics, Nishi-Beppu Hospital, Beppu (Japan)
(Received
November
26th,
1979)
Summary The activity and the activation energy (25-43°C) of acetylcholinesterase did not differ between erythrocytes from patients with Duchenne muscular dystrophy (DMD) and controls. The Hill coefficient for the inhibition of the enzyme activity by fluoride was 1 in both groups. The dose of fluoride for 50% inhibition of the enzyme activity differed between samples prepared in different ways, but not between DMD and controls. The present results suggest that there is no gross abnormality in erythrocyte acetylcholinesterase or its environment in the erythrocyte membrane in DMD.
Introduction Although often conflicting, various abnormalities of erythrocytes have been described in Duchenne muscular dystrophy (DMD). Erythrocyte membranes, especially in man, have a high activity of acetylcholinesterase (AchE), an enzyme localized on the outer side of the membrane [l]. The physiological role of this enzyme in erythrocytes has not been defined but it seems a useful probe for the study of membranes [ 2,3]. To our knowledge, there has been one study on erythrocyte AchE in DMD which described abnormal response to inhibitors [ 41. High activity of muscle AchE was reported in DMD [5]. We therefore studied the characteristics of erythrocyte AchE in DMD.
* Correspondence cine,
Kyushu
should University.
be addressed Fukuoka
to: 812,
Hid&i Japan.
Igisu,
M.D.,
Department
of Neurology.
Faculty
of Medi-
242
Subjects and methods
All subjects were males. Experiments 1 and 2 were done on separate days, but within each experiment samples from patients with DMD and age-matched controls (patients with cerebral palsy in experiment 1 and patients with asthma in experiment 2) were obtained on the same day at almost the same time (just before lunch) and processed simultaneously. In experiment 1, all subjects were in the same institution. For anticoagulation, EDTA-2Na (experiment 1) or heparin-Na (experiment 2) was used. The blood samples were immediately placed on ice until 5 h (experiment 1) or 2 h (experiment 2) later, respectively, when the preparation of the samples was started. In experiment 1, we made ghosts with a method similar to Brown et al. [6] : after plasma and buffy coat were removed, the erythrocytes were washed three times with isotonic saline. The erythrocytes were hemolyzed with 2 mmol/l Tris-HCl, pH 7.4, containing 0.02 mmol/l EDTA-2Na. The ghosts were sedimented at 20 000 X g for 20 min and washed 5 times with the same buffer. After the second and the last centrifugation, the ghosts were homogenized with a Teflon pestle. During this procedure the temperature was kept at 4°C. The ghosts, suspended in the buffer (the protein concentration approximately 5 mg/ml), were kept at -80°C until use when the protein concentration was made 2 mg/ml with 2 mmol/l Tris-HCl, pH 7.4. Then equal volumes of 0.1 mol/l phosphate buffer, pH 7.4, or the same buffer containing 2% Triton X-100 were added, making the final protein concentration of the ghost suspension 1 mg/ml, and kept on ice overnight. In experiment 2, the erythrocytes were washed twice with isotonic saline and then once with 0.1 mol/l phosphate buffer, pH 7.4. The erythrocytes were resuspended in the same buffer to make the hemoglobin concentration 4.0 g/dl, kept on ice overnight and hemolyzed with freeze and thaw in dry ice-acetone. The AchE activity was assayed by a slight modification of the method of Ellman et al. [ 71. The reaction mixture consisted of 3 ml of 0.1 mol/l phosphate buffer, pH 7.4, containing 0.25 mmol/l 5,5’dithiobis-2-nitrobenzoic acid, 50 ,YI of 40 mmol/l acetylthiocholine iodide, and 20 ~1 of ghost suspension or 10 ~1 of hemolysate. An appropriate volume (5-35 ~1) of 240 mmol/l NaF was added when necessary. Addition of 35 ~1 of H,O or 240 mmol/l NaCl caused no change in the enzyme activity. The change in absorbance at 412 nm was observed with a spectrophotometer (Hitachi 200-20) equipped with a recorder (Beckman-Toshiba U-125M). The temperature of the buffer and the cuvette holder was controlled with a circulation bath (Lauda KSRD). The protein concentration of the ghost suspension was measured by the method of Lowry et al. [8] and the hemoglobin concentration of the erythrocyte suspension by the cyanmethemoglobin method [9]. The activation energy and the Hill coefficient for the inhibition of the enzyme activity by fluoride were obtained graphically according to the following equations: lnK=-EIRT+a,
where K is the reaction rate; R is gas constant; constant; E is the activation energy.
T is absolute
temperature;
a is a
243 LN ACE~YL~HOLINESTERASE
ACTIVITY
244
TABLE
I
AchE
ACTIVITY
(25*C)
AND
ACTIVATION
ENERGY
(25-43°C)
i. SD.1
Controls
DMD
Kxpcrimen
(MEAN
t 1 (ghosts)
N
fi
5
age
c S.D.)
(mean
activity
(pmoles
Triton
14.0 thiocholine
X-100
activation
energy
hydrolized/mg
(~imoles
i 5.7
proteinlmin)
(-_)
1.96
+ 0.18
2.08
(+I
1.94
+ 0.19
2.12
+ 0.17
2.40
+ 0.27
2.22
? 0.11
(caI/molf
Experimer2 t 2 (tz~m~t~,sates~ N age (mean + S.D.) activity
12.7
-? 5.1
6
5 9.4
thiocholine
k 0.17
t
1.9
hydrolizedfg 30.2
10.8
f 0.3
30.5
+ 1.6
Hbimin)
+ 2.5
log V/(V, - V) = log &.s - n log(l) where V, is the reaction velocity without fluoride; V is the reaction velocity with fluoride; K0,5 is the fluoride concentration when V/( V,-- V) is equal to 1.0, i.e., the dose of fluoride for 50% inhibition of the enzyme activity; I is the concentration of fluoride; n is the Frill coefficient. Results The AchE activity at 25°C showed no significant difference between DMD and controls either in hemolysates or ghosts with Triton X-100, or without (Table I). The activation energy (25-43’C) of the ghost AchE obtained from the Arrhenius plot (Fig. 1) did not differ between DMD and controls (Table I). When the AchE activity (at 25°C) was plotted against the dose of NaF, a
TABLE HILL
II COEFFICIENT
FOR
THE
INHIBITION
OF
AchE
BY
FLUORIDE
AND
Kg
5 AT
S.D.) -. __..._
--
DMD -...
.__
_
Experiment Hill
I (ghosts)
coefficient Triton
k’O.5
X-100
(-_)
1.01
-1 0.04
1.00
r 0.03
(+)
1.00
-t 0.04
0.99
f 0.04
(-_)
0.97
* 0.03
0.96
(+)
0.91
f 0.02
0.91
i 0.03
0.97
t 0.04 --
0.64
(mmoI/l)
Triton
X-100
Experiment 2 (hemolysates) Hill coefficient k’o.5 Number KO.5
0.67
and age of the subjects
is the
* Different Different
dose
of fluoride
@
< 0.01)
@
< 0.001)
*
0.96
(mmoI/lf
--.--
**
-~.-_
Controls
are the same
for
from from
**
50%
the the
inhibition
Ko.5 h’O.5
without
f 0.02 *
f 0.02
i 0.04 **
+ 0.03 .-.
as in Table of
AchE
Triton
of ghosts
with
I. activity. X-100. or without
Triton
X-100.
_
25OC
(MEAN
f
12
n
1
C-I
2
3
2
3
\
1
2
2
\
2
1
3 1
l
2
l2
2
1
c-3 kL?
.5
1
1
.5
n
3
1
c-2
2
~
1
1 .5 IIY!z?
2 D-3
1
2
1
2
2
Fig, 2. Effect of N@ on ghost A&E activity. D 1-5 ad activity @moles thiocboline hydrolyzed/mg protein/min).
1
2
1 .5
2
1
‘=
\
D-2
1 .5 L.YIL 1
2
1
2
123
1
2
3
rl
\
L
\-,
1 -
2
I 1
1
2
1
1
3 mM NaF
3
2
C-5
2
h
1
1
2
3 mM NaF
C l-6 are fame as in Fig. 1. The abscissa indicates concentration of NaF (mnolfl) Inset: the same data were replotted acCWding to the Hill WUatiOD.
3
2
3
1
2
and the ordinate
AchE
246
hyperbolic curve was obtained and the controls (Fig. 2). A significant difference of Ko,s was and between ghosts with and without ence between DMD and controls (Table
Hill coefficient
was 1 in both DMD and
noted between hemolysates and ghosts Triton X-100, but there was no differII).
Discussion It was reported that the extent of suppression of the AchE activity by a single dose of inhibitors including fluoride was abnormal in erythrocytes in DMD [4]. On the other hand, the Hill coefficient for the inhibition by fluoride of erythrocyte AchE activity appears a very sensitive indicator of the membrane characteristics [lO,ll]. We therefore studied the effect of fluoride on AchE activity with various doses of the drug and did Hill plot analysis. The dose-response curve was not sigmoid and the Hill coefficient was 1 in DMD and control erythrocytes, indicating no allosteric inhibition in either group. The dose of fluoride for a 50% inhibition of AchE activity differed between hemolysates and ghosts, or between ghosts with Triton X-100 and without it, showing that difference in preparation of samples affects the extent of suppression of the enzyme activity. But no difference was found between DMD and controls. In several previous studies on erythrocytes in DMD, samples from adults were included in the controls. However, erythrocytes from children and adults differ in various aspects [ 12-141. Differences in procedures for preparing ghosts has profound effects on membrane characteristics [ 15,161, and this may be the cause of the discrepancies in the findings in erythrocytes in DMD [14, 161. We, therefore, (1) used sex- and age-matched controls, (2) obtained samples from patients and controls at almost the same time on the same day, (3) processed the samples and assayed the enzyme activity from the two groups simultaneously, and (4) examined hemolysates as well as ghosts. The activation energy of an enzyme-catalysed reaction is considered an indicator of the conformation of the active site and it may be affected by the membrane fluidity [ 171. The Hill coefficient for the inhibition of AchE by fluoride is also affected by the membrane characteristics [ 10,111. We did not see any abnormality in these parameters or in the AchE activity in erythrocytes in DMD within the temperature range we studied. Thus, there seems to be no gross abnormality in the AchE or its environment in the erythrocyte membrane in DMD. Acknowledgements We thank Dr. Hamasaki (Department of Biochemistry, School of Medicine, Fukuoka University) for valuable comments, and Dr. Nishima (MinamiFukuoka Hospital) for kind cooperation. This work was supported by grants from the Ministry of Health and Welfare, Japan.
247
References Niday,
E., Wang,
Aloni,
B. and
Jarrett,
H.W.
Goedde.
and
H.W..
(1977)
Klin.
5
Kar,
6
Brown,
7
Ellman,
C.S.
Livne,
N.C.
and
Alaupovic,
(1974)
55,
J.T.
(1976)
H.G.,
Das,
Bioehim.
Biophys,
Biophys.
Acta
Biochim.
Biophys.
P.K.,
Agarwal.
339,
Acta
469,
180-193
359-366 Acta
D.P..
448,
Lang,
314-324
H..
Wiirzburg.
U. and
Beckmann,
R.
Pharmacol.
7,
215-217
Pearson,
C.M.
(1973)
Chattopadhyay,
G.L.,
P. (1977)
Biochim.
Penniston.
Benkmann,
Wschr.
H.D.,
and A.
Courtney,
Biochem.
S.K. K.D.,
and
Med.
Pat&,
Anders.
V.
A.B. and
7. 452459 (1967)
Science
Featherstone,
157.1577-1578
R.M.
(1961)
Biochem.
88-95 8
Lowry,
9
Dacie,
O.H., J.V.
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