Vol. 67, No. 3, 1975
BIOCHEMICAL
SUPEROXIDE
DISMUTASE
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ACTIVITIES
TRISOMY
P. M.
x
SINETx,
F.
Laboratoire
de Biochimie
Necker-Enfants XX
de Biologie
13,
P. et M.
rue
A. M.
149 rue
September
75005
MICHELSONxx
and H.
Institut
IN
Hopital
PARIS
FRANCE.
Service
PARIS,
JEROMEx
de Prog&ese,
de Sevres,
Physico-Chimique, Curie,
PLATELETS
21
Genetique,
Malades,
Institut
Received
LAVELLEXX,
OF BLOOD
75730,
de Biochimie-Physique,
FRANCE.
25,1975
SUMMARY. - Mitochondrial and cytoplasmic superoxide dismutase activities have been determined in blood platelets from normal subjects and from trisomy 21 patients. The cytoplasmic enzyme (erythrocuprein) shows the same increase of 50 ‘$ in trisomy 21 compared with controls, previously noted in erythrocytes, whereas the mitochondrial manganese containing enzyme is significantly decreased by one third in platelets from cases of trisomy 21. The biological significance of these results is discussed.
We have superoxide result
dismutase
has
plasmic
since
enzyme
cells
activity
thesis
that
(SOD
- 1) containing
+ 02:
located
gene-dosage
effect.
another
of SOD,
(4, 5, 9) for enzyme These
which
properties
sion, nine
we have normal
and zinc,
+ 2 H+ ->
H202
a mitochondrial
enzyme
measurement
of a relationship
measured
sited
whereas
radicals
this
than
of the two types between
Cop.vright Q 19 75 hv Academic Press. 11x. All rights of reproduction in ary form reserved.
trisomic
supports
gene
21 patients
the hypo-
there
exists
manganese The
cuprozinc
is not (5,9).
of SOD in cell
dosage
to
21 is due to a direct
6 (10).
the manganoenzyme
cyto-
the SOD - 1
- 2) containing
on chromosome
This
according
erythrocytes,
(SOD
This
in all human
Because
finding
the SOD - 1 and SOD - 2 activities and eleven
(2, 3).
SOD in trisomy other
21 (1).
is present
+ 02 (6).
21 (7,8),
of the erythrocyte
allow
laboratories
of superoxide
by cyanide
subjects
other
copper
in cells
erythrocyte
1. 15. 1. 1. ) in trisomy
in several
the gene has been
In quest
(EC.
of human
the dismutation
However,
is inhibited
an increase
on chromosome
the increase
type
(SOD)
confirmed
: 02’-
has been
reported
been
(4, 5) and catalyses
the reaction gene
previously
and gene
extracts. expres-
in platelets
of similar
age.
from
BIOCHEMICAL
Vol. 67, No. 3, 1975
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
MATERIAL
The
platelets
described
by Pinot
of 10 mM
phosphate
Protein
The
were
in which
anions
by the xanthine
generated
assay
of the SOD activity,
NaOH
buffer
and suitable
reaction
is initiated
reaction activity
aliquots
these
mixture was
in order
obtained
The
(0. 25 and 0. 15 volumes) by centrifugation
was done by Salin
for
according
extracts
of SOD activity.
between
organic
lysates
were
and the heavy
SOD activity
3 ml).
The
(Boehringer) light
intensity
SOD activity
added
to the
activity.
SOD - 1
the two assays.
of crude
with
cold which
then
used
polyacrylamide
of Beauchamp
extracts
ethanol-chloroform occurs for
was removed electrophoresis.
gel electrophoresis
and Fridovich
(I. 5) modified
(5).
results
of human
enzyme
precipitate
after
The
10 PM hypo-
The
cyanide
treatment
mixed
superoxide
out in 0. 1 M glycine-
of maximal
RESULTS The
emission.
50 % inhibition
to the technique
and McCord
light
oxidase
any cuprozinc
-xanthine
for
of 15 ~1 of xanthine
to inhibit
des-
of the chemi-
luminol
volume
of 2 mM
(12).
previously
(final
extracts
and in presence
the different
hypoxanthine
is carried
of activity
platelets
are
AND stained presented
905
to
2 hours.
: inhibition
with
(15, 000 g) and the supernatant
Staining
method,
oxygen-
added
method
of cell
by the difference
out.
for
10 PM luminol,
gel electrophoresis,
:also carried
used
decreases
to one unit
frozen was
was
at 4” C for
is used
modified.
conditions
in absence
X-100”
100 pM EDTA,
as corresponding
For was
slightly
by injection
Under
was measured
by an indirect
oxidase
in 1 ml and was
by the Lowry
SOD in competition
pH 9, containing
xanthine
was
in the system
oxidase-luminol,
is considered
“Triton
was determined
produced
suspended M NaCl,
shaken
principle
as
and the mitochondria
and gently
the following
reaction
(1 mg/ml).
0,14
of 1 % (v/v)
measured
prepared
was
of the platelets
concentration
(13, 14) where
luminescent
pH 7, 2, containing lysis
were
pellet
(15, 000 g) the supernatant
SOD activities
cribed
blood
platelet
suspension
centrifugation
assays.
The
: one volume
of platelet
METHODS
5 ml of venous
(11).
thawing,
as follows
ten volumes After
et al. buffer,
at - 70” C. After completed
from
AND
DISCUSSION gel electrophoresis in figure
or crude
1. An identical
profile
Vol. 67, No. 3, 1975
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Platelets
Fig.
1:
Results
superoxide
glycine leucocytes
after
from
performed
isolated
human
was
1) without solvent
stained
platelets
current
carried
treatment
gels
of 4 mA of Pinot
2) with 3) without
10
et al.
-3
with
per
out as described
cyanide
for
and leucocytes.
in 7. 5 % acrylamide
by the procedure
treatment
extracts organic
gel electrophoresis
pH 8. 5 and a constant
were solvent
Crude
activity
was
buffer,
organic
of polyacrylamide
dismutase
Electrophoresis
Leucocytes
Tris-
gel. (11)
for
Human ; lysis
and
the platelets.
M cyanide
: extracts -3 4)withlO M
cyanide
cyanide.
Table
1 -
SOD activities in units/mg protein (mean + standard deviation)
Trisomy P
SOD
46. 73 + 11. 5
SOD - 2 (mitochondrial) SOD - 1 (cytoplasmic) = Total SOD minus SOD - 2
SOD activities
6.44
2 1. 65
40.30
-+ 10.90
in platelets
:
Trisomic 21 patients n = 11
Controls n=9
Total
Ratio
from
67. 10 +- 13. 52 4.31
+- 1. 70
62. 79 -+ 12. 93
normal
controls
906
Controls
LO.01
1.44
4 0.02
0. 67
4 0. 001
1. 56
and trisomic
21 patients.
21
Vol. 67, No. 3, 1975
is obtained bands
BIOCHEMICAL
with
are
platelets
present
and mitochondrial
from
or trisomic
21 subjects.
and have
Rf values
corresponding
to those
SOD from
human
is fully
both
resistant
SOD thus chicken As
being
appears liver
shown
RESEARCH COMMUNICATIONS
normal
all the SOD activity enzymes
AND BIOPHYSICAL
retained
in figure
after
(5).
the organic
which
from are
1, cyanide
other
Human
destroyed
inhibition
we found
solvent
that
treatment, mitochondrial
mitochondrial
definitely
distinct
of cytoplasmic
In addition,
to chloroform-ethanol.
to be different
enzyme,
leukocytes
Two
SODS,
such
as the
by such treatment
is observed
for
(9).
the cytoplasmic
enzyme.
The platelets
from
activities
are
finding
as their
which
have
shows
controls
values
a modified
life
and even
rable
span
with
their
increased
observed
(17).
in erythrocytes, have
been
and trisomy
21 patients
(18, 19).
Thus,
the increase
of the SOD - 1 activity
The platelets
from
mitochondrial t -isomic
by the lack
trisomy
21.
assays.
Only
gene tion oxide
Yet
of this anions.
in mitochondria
of these
the ratio
between
on
enzymes
of the average
which
in which
in platelets
SOD - 2 activity 21 subjects.
of reports the platelet
are these
is located
discerned
is sited
is quite
compa-
no differences
normal
subjects
as in the erythrocytes,
21 is most
likely
due to a
effect.
difficult
but the results
the levels
cells
in trisomy
as alkaline
which
hand,
21
in leucocytes
such
SOD - 1 is I.. 56,
characteristics
gene-dosage
However,
On the other
in maturity
direct
enzymes
This
in trisomy
Thus,
dehydrogenase
21 SOD - l/control
the ratio
21,
SOD
21 patients.
properties
span.
in
cytoplasmic
of trisomic
life
in trisomy
(17).
The
of platelet
glucose-6-phosphate
in the platelets
of SOD activity
21 patients.
in platelets
and perhaps
are
of trisomic
trisomy
to modifications (16)
unaltered
of estimations
increased
density
the X-chromosome, are
and from
be related
phosphatase
the results
significantly
might
such
table
oxidase
studied
and depend may
appear
6, raise
an increase well
occur
has been
on the type
of substrates
surprising
because
the hypothesis levels
as a consequence
907
in (20, 21, 22) used
for
the SOD - 2
of a possible
regula-
of SOD - 1 and/or
in the superoxide
in
is made
mono-amine
by the intra-cytoplasmic
could
finding
of mitochondria
which
Moreover,
of this
to the properties
on chromosome
enzyme
analysis
decreased
relating
conflicting, data,
The
is significantly
radical
of the reduced
super-
concentration SOD - 2
BIOCHEMICAL
Vol. 67, No. 3, 1975
level.
In view
(23, 24), quently
this
of the highly might
in cell
reactive
be the origin
function
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
in trisomy
and toxic of damage
properties
of these
in the mitochondria
radicals and conse-
2 1.
ACKNOWLEDGEMENTS
We are Rethore,
Dr.
des Enfants to Annie
Marguerite Malades,
Bazin
This la Recherche Conseil well
indebted
for
to Prof. Prieur
Paris, technical
for
is supported
Medicale
Francaise,
as the DGRST
of the Faculte (Contract
Lejeune,
and Prof.
Claire
allowing
Dr.
Marie-Odile
Nihoul-Fekete
us to study
their
of Hopital
patients,
and
assistance.
study
Scientifique
Jerome
by grants CNRS
from
(ERA
de Medecine
INSERM,
Fondation
pour
no 47 and ER no 103) and the Necker
- Enfants
Malades,
as
no 73 7 1183).
REFERENCES
1. 2. 3. 4. 5. 6.
Sinet, P. M., Allard, D., Lejeune, J. and Jkrbme, H. (1974) C. R. Acad. Sci. Paris 278, 3267-3270. K. and Lavelle, F. (1975) Michelson, A. M. , Puget, in preparation. Frants, R. R. , Eriksson, A. W., Jongbloet, P. H. and Hamers, A. J. (1975) Lancet, ii, 42-43. E. and Tarnvik, A. (1973) Beckman, G., Lundgren, Human Hered. 23, 338-345. Salin, M. L. anFMcCord, J. M. (1974) J. Clin. Invest. 54, 1005-1009. &Cord, J. M. and Fridovich, I. (1969) J. Biol. Chem. 244,
7. 8.
9.
248,
10. 11. 12.
6049-6055.
J. and Ruddle, F.H. (1973) Tan, Y. H. , Tischfield, J. Exp. Med. 137, 317-330. Sinet, P. M., Couturier, J., Dutrillaux, B., Poissonier, M. 0. , Allard, D., Lejeune, J. and Raoul, O., Rethore, Jerome, H. (1975) Exp. Cell. Res. in press. Weisiger, R. A. and Fridovich, I. (1973) J. Biol. Chem.
M.,
4793-4796.
F. and Ruddle, F. H. Gagan, R., Tischfield, J., Ricciuti, (1973) Humangenetik 20, 203-209. F and Jerome, H. (1970) Ann. Biol. Pinot, C., Kamoun, Clin. 28, 85-88. LowryTO. H., Rosenbrough, N. J. , Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem. 193, 265.
908
Vol. 67, No. 3, 1975
13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Lavelle, F., Durosay, P. and Michelson, A. M. (1974) Biochimie 56, 451-458. Puget, K. and Michelson, A.M. (1974) Biochimie 56, 1255-1267. Beauchamp, C. and Fridovich, I. (1971) Anal. Biochem. -’44 276-287. Kamoun, P. and Jerame, H. (1975) in : Serotonin in Mental Disorders (D. I. Boullin ed. ), John Wiley London (in press). Hsia, D. Y. Y., Justice, P., Smith, G. F. and Dowben, R. M. (1971) Amer. J. Dis. Child. 121, 153-161. Layser, R. B. and Epstein, C. J. (1972) Amer. J. Hum. Genet. 24, 533-543. qarkes, R. S. and Baughan, M.A. (1969) Amer. J. Hum. Genet. 21, 430-439. Benson, P. F. and Southgate, J. (1971) Amer. J. Hum. Genet. 23, 211-214. zasonen, M. K. , Solatuntari, E. and Kivalo, E. (1964) Psychopharmacologia 2, 120- 124. Lott, I. T. , Chase, T. N. and Murphy, D. L. (1972) Pediat. Res. 6, 730-735. Lavelle, F. , Michelson, A. M. and Dimitrijevic, L. (1973) Biochem. Biophys. Research Communs 55, 350-357. Michelson, A. M. and Buckingham, M.E. (1974) Biochem. Biophys. Research Communs -58, 1079-1086.
909
BIOCHEMICAL
SUPEROXIDE
DISMUTASE
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ACTIVITIES
TRISOMY
P. M.
x
SINETx,
F.
Laboratoire
de Biochimie
Necker-Enfants XX
de Biologie
13,
P. et M.
rue
A. M.
149 rue
September
75005
MICHELSONxx
and H.
Institut
IN
Hopital
PARIS
FRANCE.
Service
PARIS,
JEROMEx
de Prog&ese,
de Sevres,
Physico-Chimique, Curie,
PLATELETS
21
Genetique,
Malades,
Institut
Received
LAVELLEXX,
OF BLOOD
75730,
de Biochimie-Physique,
FRANCE.
25,1975
SUMMARY. - Mitochondrial and cytoplasmic superoxide dismutase activities have been determined in blood platelets from normal subjects and from trisomy 21 patients. The cytoplasmic enzyme (erythrocuprein) shows the same increase of 50 ‘$ in trisomy 21 compared with controls, previously noted in erythrocytes, whereas the mitochondrial manganese containing enzyme is significantly decreased by one third in platelets from cases of trisomy 21. The biological significance of these results is discussed.
We have superoxide result
dismutase
has
plasmic
since
enzyme
cells
activity
thesis
that
(SOD
- 1) containing
+ 02:
located
gene-dosage
effect.
another
of SOD,
(4, 5, 9) for enzyme These
which
properties
sion, nine
we have normal
and zinc,
+ 2 H+ ->
H202
a mitochondrial
enzyme
measurement
of a relationship
measured
sited
whereas
radicals
this
than
of the two types between
Cop.vright Q 19 75 hv Academic Press. 11x. All rights of reproduction in ary form reserved.
trisomic
supports
gene
21 patients
the hypo-
there
exists
manganese The
cuprozinc
is not (5,9).
of SOD in cell
dosage
to
21 is due to a direct
6 (10).
the manganoenzyme
cyto-
the SOD - 1
- 2) containing
on chromosome
This
according
erythrocytes,
(SOD
This
in all human
Because
finding
the SOD - 1 and SOD - 2 activities and eleven
(2, 3).
SOD in trisomy other
21 (1).
is present
+ 02 (6).
21 (7,8),
of the erythrocyte
allow
laboratories
of superoxide
by cyanide
subjects
other
copper
in cells
erythrocyte
1. 15. 1. 1. ) in trisomy
in several
the gene has been
In quest
(EC.
of human
the dismutation
However,
is inhibited
an increase
on chromosome
the increase
type
(SOD)
confirmed
: 02’-
has been
reported
been
(4, 5) and catalyses
the reaction gene
previously
and gene
extracts. expres-
in platelets
of similar
age.
from
BIOCHEMICAL
Vol. 67, No. 3, 1975
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
MATERIAL
The
platelets
described
by Pinot
of 10 mM
phosphate
Protein
The
were
in which
anions
by the xanthine
generated
assay
of the SOD activity,
NaOH
buffer
and suitable
reaction
is initiated
reaction activity
aliquots
these
mixture was
in order
obtained
The
(0. 25 and 0. 15 volumes) by centrifugation
was done by Salin
for
according
extracts
of SOD activity.
between
organic
lysates
were
and the heavy
SOD activity
3 ml).
The
(Boehringer) light
intensity
SOD activity
added
to the
activity.
SOD - 1
the two assays.
of crude
with
cold which
then
used
polyacrylamide
of Beauchamp
extracts
ethanol-chloroform occurs for
was removed electrophoresis.
gel electrophoresis
and Fridovich
(I. 5) modified
(5).
results
of human
enzyme
precipitate
after
The
10 PM hypo-
The
cyanide
treatment
mixed
superoxide
out in 0. 1 M glycine-
of maximal
RESULTS The
emission.
50 % inhibition
to the technique
and McCord
light
oxidase
any cuprozinc
-xanthine
for
of 15 ~1 of xanthine
to inhibit
des-
of the chemi-
luminol
volume
of 2 mM
(12).
previously
(final
extracts
and in presence
the different
hypoxanthine
is carried
of activity
platelets
are
AND stained presented
905
to
2 hours.
: inhibition
with
(15, 000 g) and the supernatant
Staining
method,
oxygen-
added
method
of cell
by the difference
out.
for
10 PM luminol,
gel electrophoresis,
:also carried
used
decreases
to one unit
frozen was
was
at 4” C for
is used
modified.
conditions
in absence
X-100”
100 pM EDTA,
as corresponding
For was
slightly
by injection
Under
was measured
by an indirect
oxidase
in 1 ml and was
by the Lowry
SOD in competition
pH 9, containing
xanthine
was
in the system
oxidase-luminol,
is considered
“Triton
was determined
produced
suspended M NaCl,
shaken
principle
as
and the mitochondria
and gently
the following
reaction
(1 mg/ml).
0,14
of 1 % (v/v)
measured
prepared
was
of the platelets
concentration
(13, 14) where
luminescent
pH 7, 2, containing lysis
were
pellet
(15, 000 g) the supernatant
SOD activities
cribed
blood
platelet
suspension
centrifugation
assays.
The
: one volume
of platelet
METHODS
5 ml of venous
(11).
thawing,
as follows
ten volumes After
et al. buffer,
at - 70” C. After completed
from
AND
DISCUSSION gel electrophoresis in figure
or crude
1. An identical
profile
Vol. 67, No. 3, 1975
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Platelets
Fig.
1:
Results
superoxide
glycine leucocytes
after
from
performed
isolated
human
was
1) without solvent
stained
platelets
current
carried
treatment
gels
of 4 mA of Pinot
2) with 3) without
10
et al.
-3
with
per
out as described
cyanide
for
and leucocytes.
in 7. 5 % acrylamide
by the procedure
treatment
extracts organic
gel electrophoresis
pH 8. 5 and a constant
were solvent
Crude
activity
was
buffer,
organic
of polyacrylamide
dismutase
Electrophoresis
Leucocytes
Tris-
gel. (11)
for
Human ; lysis
and
the platelets.
M cyanide
: extracts -3 4)withlO M
cyanide
cyanide.
Table
1 -
SOD activities in units/mg protein (mean + standard deviation)
Trisomy P
SOD
46. 73 + 11. 5
SOD - 2 (mitochondrial) SOD - 1 (cytoplasmic) = Total SOD minus SOD - 2
SOD activities
6.44
2 1. 65
40.30
-+ 10.90
in platelets
:
Trisomic 21 patients n = 11
Controls n=9
Total
Ratio
from
67. 10 +- 13. 52 4.31
+- 1. 70
62. 79 -+ 12. 93
normal
controls
906
Controls
LO.01
1.44
4 0.02
0. 67
4 0. 001
1. 56
and trisomic
21 patients.
21
Vol. 67, No. 3, 1975
is obtained bands
BIOCHEMICAL
with
are
platelets
present
and mitochondrial
from
or trisomic
21 subjects.
and have
Rf values
corresponding
to those
SOD from
human
is fully
both
resistant
SOD thus chicken As
being
appears liver
shown
RESEARCH COMMUNICATIONS
normal
all the SOD activity enzymes
AND BIOPHYSICAL
retained
in figure
after
(5).
the organic
which
from are
1, cyanide
other
Human
destroyed
inhibition
we found
solvent
that
treatment, mitochondrial
mitochondrial
definitely
distinct
of cytoplasmic
In addition,
to chloroform-ethanol.
to be different
enzyme,
leukocytes
Two
SODS,
such
as the
by such treatment
is observed
for
(9).
the cytoplasmic
enzyme.
The platelets
from
activities
are
finding
as their
which
have
shows
controls
values
a modified
life
and even
rable
span
with
their
increased
observed
(17).
in erythrocytes, have
been
and trisomy
21 patients
(18, 19).
Thus,
the increase
of the SOD - 1 activity
The platelets
from
mitochondrial t -isomic
by the lack
trisomy
21.
assays.
Only
gene tion oxide
Yet
of this anions.
in mitochondria
of these
the ratio
between
on
enzymes
of the average
which
in which
in platelets
SOD - 2 activity 21 subjects.
of reports the platelet
are these
is located
discerned
is sited
is quite
compa-
no differences
normal
subjects
as in the erythrocytes,
21 is most
likely
due to a
effect.
difficult
but the results
the levels
cells
in trisomy
as alkaline
which
hand,
21
in leucocytes
such
SOD - 1 is I.. 56,
characteristics
gene-dosage
However,
On the other
in maturity
direct
enzymes
This
in trisomy
Thus,
dehydrogenase
21 SOD - l/control
the ratio
21,
SOD
21 patients.
properties
span.
in
cytoplasmic
of trisomic
life
in trisomy
(17).
The
of platelet
glucose-6-phosphate
in the platelets
of SOD activity
21 patients.
in platelets
and perhaps
are
of trisomic
trisomy
to modifications (16)
unaltered
of estimations
increased
density
the X-chromosome, are
and from
be related
phosphatase
the results
significantly
might
such
table
oxidase
studied
and depend may
appear
6, raise
an increase well
occur
has been
on the type
of substrates
surprising
because
the hypothesis levels
as a consequence
907
in (20, 21, 22) used
for
the SOD - 2
of a possible
regula-
of SOD - 1 and/or
in the superoxide
in
is made
mono-amine
by the intra-cytoplasmic
could
finding
of mitochondria
which
Moreover,
of this
to the properties
on chromosome
enzyme
analysis
decreased
relating
conflicting, data,
The
is significantly
radical
of the reduced
super-
concentration SOD - 2
BIOCHEMICAL
Vol. 67, No. 3, 1975
level.
In view
(23, 24), quently
this
of the highly might
in cell
reactive
be the origin
function
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
in trisomy
and toxic of damage
properties
of these
in the mitochondria
radicals and conse-
2 1.
ACKNOWLEDGEMENTS
We are Rethore,
Dr.
des Enfants to Annie
Marguerite Malades,
Bazin
This la Recherche Conseil well
indebted
for
to Prof. Prieur
Paris, technical
for
is supported
Medicale
Francaise,
as the DGRST
of the Faculte (Contract
Lejeune,
and Prof.
Claire
allowing
Dr.
Marie-Odile
Nihoul-Fekete
us to study
their
of Hopital
patients,
and
assistance.
study
Scientifique
Jerome
by grants CNRS
from
(ERA
de Medecine
INSERM,
Fondation
pour
no 47 and ER no 103) and the Necker
- Enfants
Malades,
as
no 73 7 1183).
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