Vol. 67, No. 2, 1975
BIOCHEMICAL
PURIFICATION
AND PROPERTIES OF CYSTATHIONINE N. Tudball
Department
of Biochemistry,
Received
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
September
and Maureen University
SYNTHASE FROM HUMAN LIVER
A. Reed
College,
Cathays
Park,
Cardiff
CFl XL,
U.K.
3,1975 Summary
Cystathionine enzymically 250,000
g-synthase
active
forms.
Both
and are dependent
has been
isolated
enzymes,
a and@possess
upon pyridoxal
from human liver
phosphate
in two
a molecular
weight
of
as a cofactor.
Introduction Cystathionine EC4.2.1221
is
g-synthase
a key enzyme
normal
methionine
reported
the
which
(deaminating),
EC4.2.1.131
Kashiwamata
evidence
entities.
are properties In humans,
for
the condition
report
(Porter,
preparation enzyme is properties
activity.
More
showed
that
Grishaver
Braunstein
(1965)
hydro-lyase
on purified
rat-liver
and Kimura,
synthase
1968;
and dehydratase
(1971)
synthase
synthase
&serine
Nakagawa
--et al.
deficiency
were
have now presented
and serine
of cystathionine
as homocystinuria and Jones,
The present
of cystathionine
with
sulphydrase
of the same protein.
of the enzyme obtained lacking.
work
the
cystathionine
a congenital known
that
associated
of cystathionine
recent 1971;
homocysteine),
and Greenberg
debydratase
and Gordon,
However,
indicates
preparation
gserine
1970)
pathway
Nagabhushanam
possessed
(Brown
(adding
transsulphuration
in mammals.
and Greenberg,
which
activities
also
synthase
two distinct
hydro-lyase
of a homogeneous
from rat-liver
cystathionine
in the
metabolism
isolation
&-serine
(Mudd --et al. 1974)
from
dealing
foetal
communication
synthase
from
with
tissue, deals
adult
synthase 1964).
Apart
a partially information
with
is
responsib
from one purified on the human
the purification
and
human liver.
Methods Routine Cop.vrighr All rights
estimation
of protein
o 19 75 by Academic Press. Inc. of reproduction in any form reserved.
was carried 550
out by the method
of Lowry
le
Vol. 67, No. 2, 1975
et al. --
(1951).
Christian
For purified
(1941)
(adding
Mudd --et al.
(1965).
of
employed.
EC4.2.1.221
The isoelectric
focusing
the method
technique
Experiments
of Warburg
8-synthase was measured
focusing
(1966).
column
enzyme,
Cystathionine
homocysteine),
and Svensson
isoelectric
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
preparations
was also
hydro-lyase
Vesterberg
BIOCHEMICAL
were
[&-serine by the method
used was that
performed
and
using
of
of
a llOm1
(L.K.B.).
Results Purification purification
steps
Stage frozen
of the
1.
were
were
O.lg-tris-hydrochloric
Any fat
through
a fine Stage
removed
to pH7.0
changes material the
3.
(total
vol
same buffer.
with
which
in a Waring
(containing
added
successive
portions concentrations
dialysed
for
had previously
been
Blender
to give
at 18,000g
the
from
with
a 25% (w/v)
for
supernatant
stage
30min
and the
was removed
I was carefully
acid. for
sufficient phosphate suspension. supernatant
by filtering
adjusted
Precipitated
1Omin.
2 preparation
to a column
material
The clear
to pH5.5
by
was immediately
supernatant
was dialysed
for
was quickly
was washed
three
The dialysed
with
pH6.5
starting
synthase
of the
of 0.12,
24h against
starting
0.2g changes
eluates
buffer
and 0.3M. = (total from
551
buffer
activity
Stage
with
the eluate
was eluted
by washing
containing
15&?,) of 3 were
equilibrated
until
potassium
The eluates vol
two
and the nondiffusable
(6cm x 30cm) of DEAE-cellulose
Cystathionine
24h against
and
2g-NaOH.
(65Oml)
at final
extraction
mesh.
The column
from protein.
all
EDTA and pyridoxal
no!?,> of O.O8g-K2HP04-KH2P04-buffer
was free
4.
pH8.3
at 18,000g
The Stage
(725ml)
Stage
buffer
of 0.22-acetic
with
stated
(25Og)
and macerated
The supernatant
addition
Stage
of liver
was centrifuged
nylon
by centrifuging
adjusted
samples
associated
(140~) 2.
the dropwise
otherwise
of 10m3g and 10B4g respectively)
suspension
collected.
Unless
out at 4'C.
minced acid
concns.
The resulting
carried
Post-mortem
and thawed,
at final
enzyme.
were starting
added
chloride
combined buffer.
to a column
and
with
Vol. 67, No. 2, 1975
BIOCHEMICAL
(5cm x 26cm) of DEAE-cellulose and the column with
washed
a linear
synthase
and (nos 48-61) concentrated U.S.A)
which 5.
filtration
column acid
were
subsequently
The column
and the
eluate
was developed
were
lmg protein/ml.
pooled
At this
collected
both
possessing
fractions
in
(nos separately
(Amicon
Corp.,
Lexington,
containing
23-26) and
4-5 mg
with
phosphate the
separately
4 preparations
equilibrated
were
same buffer Those
to
applied
to a
concentration
at a flow fractions
rate
of
of 3Oml/h
containing
to yield
the respective
subjected
O.lM-Tris-hydrochloric =
at a final
and concentrated stage
being
pooled
4 were
in lOm1 fractions.
of the enzyme activity approx.
with
was eluted
were
solutions
Stage
pyridoxal
pH6.5
at -2O'C.
The Stage G-200
8,
The active
to yield
stored
G-200.
containing
collected
eluate
~1 and
apparatus
a and S from
(5cm x 76cm) of Sephadex
10-4M.=
the
obtained.
ultrafiltration
Fractions
pH7.5
gradient,
Enzyme activity
to cx and B respectively
on Sephadex
buffer,
O.O8M-K2HP04-KH2P04-buffer
designated
an KM-50 membrane
were
RESEARCH COMMUNICATIONS
of the same buffer.
fractions
corresponding
using
with
chloride
activity
in a Diaflo
Stage gel
750ml
Two separate
cystathionine
protein/ml
equilibrated
0-0.5g-potassium
lOm1 fractions.
Mass.,
with
AND BIOPHYSICAL
most
solutions
preparations
of
were
stored
at
-2oOc. Stage yield
6.
Each enzyme preparation
solutions
containing
to isoelectric to proceed fractions protein
focusing for
48h,
(1.5ml) and pH.
the power
(2.5cm
x 40cm)
previously then
stored
input
of Sephadex
acid
buffer,
of 10-k. described at -2O'C.
3-10.
kept
at 1.5W.
removed
(Vesterberg,
~117.5 containing The resulting
to yield
fraction
corresponding
G-50
solutions
5 was concentrated
pH range
being
and sucrose
Stage
and the resulting
and each
The fractions
and the Ampholine
concentration
Ampholine
collected
pooled
hydrochloric
2.5mg protein/ml using
were
from
by gel
filtration
1969)
equilibrated
containing
552
derived
was allowed
At the end of this
assayed
for
protein
were
on a column with
were
from
period
enzyme activity,
active
phosphate
approx.
to
subjected
The separation
enzyme preparations
The enzyme preparation
solution
to enzymically
pyridoxal
further
O.lg-Tris-
at a final concentrated
lmg protein/ml fraction
as and
a possessed
Vol. 67, No. 2, 1975
an isoelectric
point
isoelectric
point
Table
of pH5.95.
of pH5.75.
enzyme and S-enzyme
Properties
BIOCHEMICAL
That These
derived
from
preparations
fraction
f3 possessed
have now been
designated
an the
a
respectively.
1 shows the
results
of a typical
of human cystathionine The molecular
filtration
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
weights
on a column
of
purification.
synthase of enzymes
Sephadex
a and B were
G-200
(1.5xm
with
1
of Cystathionine
Total volume (ml)
by gel
x 90cm) equilibrated
Table Purification
determined
S-Synthase
Total protein (md
from Human Liver
Total activity (units)*
Specific activity (units/mg protein)
Recovery
Supernatant
725
15,950
561.8
0.035
100
pH5.5
680
10,120
460.6
0.046
82
treatment
Stepwise cellulose
elution
Gradient cellulose
elution
on DEAE1900
360
387.6
1.078
69
on DEAE-
Peak c1 (Nos.
23-26)
20
87
191.1
2.197
34
Peak f? (Nos.
48-61)
20
115
163.2
1.419
29
Gel-filtration G-200
on Sephadex
Peak CI
12
12.2
151.4
12.39
26.9
Peak B
13
12.0
141.1
11.76
25.1
Peak (Y
3
2.4
64.6
26.78
11.5
Peak B
3
2.2
57.3
25.90
10.2
Isoelectric
* 1 unit
focusing
of activity
is defined
as that
amount
553
producing
lpmole
of cystathionine/2h.
Vol. 67, No. 2, 1975
O.lg
Tris-HCl
BIOCHEMICAL
buffer,
preparations
~118.3.
of cytochrome
fibrinogen
a value
Kimura,
Both
which
@enzymes,
low32
for
greater
enzymes
has been
column
was carried
ovalbumin,
albumin,
possessed
molecular
weights
for
rat-liver
enzyme
recorded
with
whereas
affinities
1970).
respect
the Km value
for
both
and Kimura,
the corresponding
the
curve pH7.9-8.3.
Antibody
prepared
its
for
out
using
aldolase, of approximately
Both After
for
against
(Nakagawa
and
with
NaBH4,
enzymes
392nm were
were
observed.
phosphate
being
conclusions
These
bound
information
afforded
by resolution
dialysis
against
of this
treatment
respective the presence
initial
according
concerning experiments.
L-cysteine
activities.
of pyridoxal of enzyme a and
the
Preparations
51% whereas Preincubation
phosphate
(final
554
then
10B4MJ for their
(1965)
with
of the at
the enzyme. phosphate
enzymes
were
(1971).
was
resolved As a result
57% of their activities 3Omin,
respective
a
of pyridoxal
of pyridoxal
and Gordon
buffer
new maxima
the rat-liver
of the resulting
6 to 89% and 92% of
phosphate
spectra
agrees
enzyme B lost
cont.
inhibition
indicative
of both
by Brown
enzyme a,
of Churchich
emission
were
involvement
whereas
with
relative
at 325nm,
for
and Gordon,
enzyme f3.
base which
(1970)
as described
enzyme a lost
When the
system
of maximum activity
at 41Onm in 0.08g
characteristics
and Greenberg
by its
x possess
at pH8.25
region
with
excitation
Schiff
Brown
sharply
to the method
at 325nm.
following spectral
obtained maxima
as a protonated
of Kashiwamata
Further
activities
examined
1969;
cross-reactive
(as judged
absorption
new maximum in each case was found reduced
rat
corresponding
a broad
50% that
was 3.98
the
enzyme b was also
than
possessed
reduction
does
the a
thus
enzyme B peaked
respect
both
The human enzymes
and Greenberg,
the
in this
was less
enzymes
enzyme 8. than
for
to homocysteine
enzyme B exhibited
effectiveness
of enzyme activity)
for
was 0.91x10-3E
respect
Kashiwamata
curve
the range
pH7.5.
with
substrates
1968;
The pH activity
however
to A-serine
enzyme a and 3.41x10B3g
(Nakagawa
over
of the
RESEARCH COMMUNICATIONS
1968). The Km value
and
Calibration
C, chymotrypsinogen,
and ferritin.
250,000,
AND BIOPHYSICAL
raised initial
in the activities.
by
BIOCHEMICAL
Vol. 67, No. 2, 1975
The above properties with
the known
of human liver
properties
seemsto
possess
progress
which
of the
two distinct are
aimed
AND BIOPHYSICAL
cystathionine
rat-liver
synthase
8-synthase Nonetheless,
system. activities.
at defining
RESEARCH COMMUNICATIONS
Further of these
the nature
are
in accord
the human liver
investigations activities
are
in
more precisely.
Acknowledgements This
work
was supported
by a grant
from the
Mental
Health
Research
Fund.
References Braunstein, Yefremova,
A.E., L.L.,
Brown,
and Gordon,
F.C.
Churchich,
J.E.
Kashiwamata, Lowry, 193,
Goryachenkova, E.V., Tolosa, E.V., Willhardt, (1971) Biochim. Biophys. Acta, 242, 247-260.
(1965)
P.H.
(1971)
Biochemistry,
S. and Greenberg,
O.H., Rosebrough, 265-275.
Mudd, S.H., 1443-1445.
Can. J. 2,
D.M.
N.J.
Farr,
Biochem.
2,
I.H.
and
484-491.
1405-1410.
(1970) A.L.
Biochim.
Biophys.
and Randall,
Acta,
R.J.
(1951)
212, J.
488-500.
biol.
Chem.
Finkelstein,
J.D.,
Irreverre,
F. and Laster,
L.
(1964)
Science,
143,
Mudd, S.H., Finkelstein, 240, 4382-4392.
J.D.,
Irreverre,
F. and Laster,
L.
(1965)
J. biol.
Chem.
Nagabhushanam, Nakagawa, Porter, 364,
H. and Kimura, P.N., Grishaver, 128-139.
Vesterberg, Warburg,
A. and Greenberg, H. (1968) M.S.
0. and Svensson, 0. and Christian,
D.M.
(1965)
Biochim.
and Jones, H. (1966)
W. (1941)
J. biol.
Chem. 240,
Biophys. O.W.
Acta, Biochem.
555
Res.
(1974) them.
Commun. 32,
Biochim. stand.
Z. 3&,
3002-3008.
0,
384-421.
Biophys. 820-834.
208-214. Acta,
BIOCHEMICAL
PURIFICATION
AND PROPERTIES OF CYSTATHIONINE N. Tudball
Department
of Biochemistry,
Received
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
September
and Maureen University
SYNTHASE FROM HUMAN LIVER
A. Reed
College,
Cathays
Park,
Cardiff
CFl XL,
U.K.
3,1975 Summary
Cystathionine enzymically 250,000
g-synthase
active
forms.
Both
and are dependent
has been
isolated
enzymes,
a and@possess
upon pyridoxal
from human liver
phosphate
in two
a molecular
weight
of
as a cofactor.
Introduction Cystathionine EC4.2.1221
is
g-synthase
a key enzyme
normal
methionine
reported
the
which
(deaminating),
EC4.2.1.131
Kashiwamata
evidence
entities.
are properties In humans,
for
the condition
report
(Porter,
preparation enzyme is properties
activity.
More
showed
that
Grishaver
Braunstein
(1965)
hydro-lyase
on purified
rat-liver
and Kimura,
synthase
1968;
and dehydratase
(1971)
synthase
synthase
&serine
Nakagawa
--et al.
deficiency
were
have now presented
and serine
of cystathionine
as homocystinuria and Jones,
The present
of cystathionine
with
sulphydrase
of the same protein.
of the enzyme obtained lacking.
work
the
cystathionine
a congenital known
that
associated
of cystathionine
recent 1971;
homocysteine),
and Greenberg
debydratase
and Gordon,
However,
indicates
preparation
gserine
1970)
pathway
Nagabhushanam
possessed
(Brown
(adding
transsulphuration
in mammals.
and Greenberg,
which
activities
also
synthase
two distinct
hydro-lyase
of a homogeneous
from rat-liver
cystathionine
in the
metabolism
isolation
&-serine
(Mudd --et al. 1974)
from
dealing
foetal
communication
synthase
from
with
tissue, deals
adult
synthase 1964).
Apart
a partially information
with
is
responsib
from one purified on the human
the purification
and
human liver.
Methods Routine Cop.vrighr All rights
estimation
of protein
o 19 75 by Academic Press. Inc. of reproduction in any form reserved.
was carried 550
out by the method
of Lowry
le
Vol. 67, No. 2, 1975
et al. --
(1951).
Christian
For purified
(1941)
(adding
Mudd --et al.
(1965).
of
employed.
EC4.2.1.221
The isoelectric
focusing
the method
technique
Experiments
of Warburg
8-synthase was measured
focusing
(1966).
column
enzyme,
Cystathionine
homocysteine),
and Svensson
isoelectric
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
preparations
was also
hydro-lyase
Vesterberg
BIOCHEMICAL
were
[&-serine by the method
used was that
performed
and
using
of
of
a llOm1
(L.K.B.).
Results Purification purification
steps
Stage frozen
of the
1.
were
were
O.lg-tris-hydrochloric
Any fat
through
a fine Stage
removed
to pH7.0
changes material the
3.
(total
vol
same buffer.
with
which
in a Waring
(containing
added
successive
portions concentrations
dialysed
for
had previously
been
Blender
to give
at 18,000g
the
from
with
a 25% (w/v)
for
supernatant
stage
30min
and the
was removed
I was carefully
acid. for
sufficient phosphate suspension. supernatant
by filtering
adjusted
Precipitated
1Omin.
2 preparation
to a column
material
The clear
to pH5.5
by
was immediately
supernatant
was dialysed
for
was quickly
was washed
three
The dialysed
with
pH6.5
starting
synthase
of the
of 0.12,
24h against
starting
0.2g changes
eluates
buffer
and 0.3M. = (total from
551
buffer
activity
Stage
with
the eluate
was eluted
by washing
containing
15&?,) of 3 were
equilibrated
until
potassium
The eluates vol
two
and the nondiffusable
(6cm x 30cm) of DEAE-cellulose
Cystathionine
24h against
and
2g-NaOH.
(65Oml)
at final
extraction
mesh.
The column
from protein.
all
EDTA and pyridoxal
no!?,> of O.O8g-K2HP04-KH2P04-buffer
was free
4.
pH8.3
at 18,000g
The Stage
(725ml)
Stage
buffer
of 0.22-acetic
with
stated
(25Og)
and macerated
The supernatant
addition
Stage
of liver
was centrifuged
nylon
by centrifuging
adjusted
samples
associated
(140~) 2.
the dropwise
otherwise
of 10m3g and 10B4g respectively)
suspension
collected.
Unless
out at 4'C.
minced acid
concns.
The resulting
carried
Post-mortem
and thawed,
at final
enzyme.
were starting
added
chloride
combined buffer.
to a column
and
with
Vol. 67, No. 2, 1975
BIOCHEMICAL
(5cm x 26cm) of DEAE-cellulose and the column with
washed
a linear
synthase
and (nos 48-61) concentrated U.S.A)
which 5.
filtration
column acid
were
subsequently
The column
and the
eluate
was developed
were
lmg protein/ml.
pooled
At this
collected
both
possessing
fractions
in
(nos separately
(Amicon
Corp.,
Lexington,
containing
23-26) and
4-5 mg
with
phosphate the
separately
4 preparations
equilibrated
were
same buffer Those
to
applied
to a
concentration
at a flow fractions
rate
of
of 3Oml/h
containing
to yield
the respective
subjected
O.lM-Tris-hydrochloric =
at a final
and concentrated stage
being
pooled
4 were
in lOm1 fractions.
of the enzyme activity approx.
with
was eluted
were
solutions
Stage
pyridoxal
pH6.5
at -2O'C.
The Stage G-200
8,
The active
to yield
stored
G-200.
containing
collected
eluate
~1 and
apparatus
a and S from
(5cm x 76cm) of Sephadex
10-4M.=
the
obtained.
ultrafiltration
Fractions
pH7.5
gradient,
Enzyme activity
to cx and B respectively
on Sephadex
buffer,
O.O8M-K2HP04-KH2P04-buffer
designated
an KM-50 membrane
were
RESEARCH COMMUNICATIONS
of the same buffer.
fractions
corresponding
using
with
chloride
activity
in a Diaflo
Stage gel
750ml
Two separate
cystathionine
protein/ml
equilibrated
0-0.5g-potassium
lOm1 fractions.
Mass.,
with
AND BIOPHYSICAL
most
solutions
preparations
of
were
stored
at
-2oOc. Stage yield
6.
Each enzyme preparation
solutions
containing
to isoelectric to proceed fractions protein
focusing for
48h,
(1.5ml) and pH.
the power
(2.5cm
x 40cm)
previously then
stored
input
of Sephadex
acid
buffer,
of 10-k. described at -2O'C.
3-10.
kept
at 1.5W.
removed
(Vesterberg,
~117.5 containing The resulting
to yield
fraction
corresponding
G-50
solutions
5 was concentrated
pH range
being
and sucrose
Stage
and the resulting
and each
The fractions
and the Ampholine
concentration
Ampholine
collected
pooled
hydrochloric
2.5mg protein/ml using
were
from
by gel
filtration
1969)
equilibrated
containing
552
derived
was allowed
At the end of this
assayed
for
protein
were
on a column with
were
from
period
enzyme activity,
active
phosphate
approx.
to
subjected
The separation
enzyme preparations
The enzyme preparation
solution
to enzymically
pyridoxal
further
O.lg-Tris-
at a final concentrated
lmg protein/ml fraction
as and
a possessed
Vol. 67, No. 2, 1975
an isoelectric
point
isoelectric
point
Table
of pH5.95.
of pH5.75.
enzyme and S-enzyme
Properties
BIOCHEMICAL
That These
derived
from
preparations
fraction
f3 possessed
have now been
designated
an the
a
respectively.
1 shows the
results
of a typical
of human cystathionine The molecular
filtration
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
weights
on a column
of
purification.
synthase of enzymes
Sephadex
a and B were
G-200
(1.5xm
with
1
of Cystathionine
Total volume (ml)
by gel
x 90cm) equilibrated
Table Purification
determined
S-Synthase
Total protein (md
from Human Liver
Total activity (units)*
Specific activity (units/mg protein)
Recovery
Supernatant
725
15,950
561.8
0.035
100
pH5.5
680
10,120
460.6
0.046
82
treatment
Stepwise cellulose
elution
Gradient cellulose
elution
on DEAE1900
360
387.6
1.078
69
on DEAE-
Peak c1 (Nos.
23-26)
20
87
191.1
2.197
34
Peak f? (Nos.
48-61)
20
115
163.2
1.419
29
Gel-filtration G-200
on Sephadex
Peak CI
12
12.2
151.4
12.39
26.9
Peak B
13
12.0
141.1
11.76
25.1
Peak (Y
3
2.4
64.6
26.78
11.5
Peak B
3
2.2
57.3
25.90
10.2
Isoelectric
* 1 unit
focusing
of activity
is defined
as that
amount
553
producing
lpmole
of cystathionine/2h.
Vol. 67, No. 2, 1975
O.lg
Tris-HCl
BIOCHEMICAL
buffer,
preparations
~118.3.
of cytochrome
fibrinogen
a value
Kimura,
Both
which
@enzymes,
low32
for
greater
enzymes
has been
column
was carried
ovalbumin,
albumin,
possessed
molecular
weights
for
rat-liver
enzyme
recorded
with
whereas
affinities
1970).
respect
the Km value
for
both
and Kimura,
the corresponding
the
curve pH7.9-8.3.
Antibody
prepared
its
for
out
using
aldolase, of approximately
Both After
for
against
(Nakagawa
and
with
NaBH4,
enzymes
392nm were
were
observed.
phosphate
being
conclusions
These
bound
information
afforded
by resolution
dialysis
against
of this
treatment
respective the presence
initial
according
concerning experiments.
L-cysteine
activities.
of pyridoxal of enzyme a and
the
Preparations
51% whereas Preincubation
phosphate
(final
554
then
10B4MJ for their
(1965)
with
of the at
the enzyme. phosphate
enzymes
were
(1971).
was
resolved As a result
57% of their activities 3Omin,
respective
a
of pyridoxal
of pyridoxal
and Gordon
buffer
new maxima
the rat-liver
of the resulting
6 to 89% and 92% of
phosphate
spectra
agrees
enzyme B lost
cont.
inhibition
indicative
of both
by Brown
enzyme a,
of Churchich
emission
were
involvement
whereas
with
relative
at 325nm,
for
and Gordon,
enzyme f3.
base which
(1970)
as described
enzyme a lost
When the
system
of maximum activity
at 41Onm in 0.08g
characteristics
and Greenberg
by its
x possess
at pH8.25
region
with
excitation
Schiff
Brown
sharply
to the method
at 325nm.
following spectral
obtained maxima
as a protonated
of Kashiwamata
Further
activities
examined
1969;
cross-reactive
(as judged
absorption
new maximum in each case was found reduced
rat
corresponding
a broad
50% that
was 3.98
the
enzyme b was also
than
possessed
reduction
does
the a
thus
enzyme B peaked
respect
both
The human enzymes
and Greenberg,
the
in this
was less
enzymes
enzyme 8. than
for
to homocysteine
enzyme B exhibited
effectiveness
of enzyme activity)
for
was 0.91x10-3E
respect
Kashiwamata
curve
the range
pH7.5.
with
substrates
1968;
The pH activity
however
to A-serine
enzyme a and 3.41x10B3g
(Nakagawa
over
of the
RESEARCH COMMUNICATIONS
1968). The Km value
and
Calibration
C, chymotrypsinogen,
and ferritin.
250,000,
AND BIOPHYSICAL
raised initial
in the activities.
by
BIOCHEMICAL
Vol. 67, No. 2, 1975
The above properties with
the known
of human liver
properties
seemsto
possess
progress
which
of the
two distinct are
aimed
AND BIOPHYSICAL
cystathionine
rat-liver
synthase
8-synthase Nonetheless,
system. activities.
at defining
RESEARCH COMMUNICATIONS
Further of these
the nature
are
in accord
the human liver
investigations activities
are
in
more precisely.
Acknowledgements This
work
was supported
by a grant
from the
Mental
Health
Research
Fund.
References Braunstein, Yefremova,
A.E., L.L.,
Brown,
and Gordon,
F.C.
Churchich,
J.E.
Kashiwamata, Lowry, 193,
Goryachenkova, E.V., Tolosa, E.V., Willhardt, (1971) Biochim. Biophys. Acta, 242, 247-260.
(1965)
P.H.
(1971)
Biochemistry,
S. and Greenberg,
O.H., Rosebrough, 265-275.
Mudd, S.H., 1443-1445.
Can. J. 2,
D.M.
N.J.
Farr,
Biochem.
2,
I.H.
and
484-491.
1405-1410.
(1970) A.L.
Biochim.
Biophys.
and Randall,
Acta,
R.J.
(1951)
212, J.
488-500.
biol.
Chem.
Finkelstein,
J.D.,
Irreverre,
F. and Laster,
L.
(1964)
Science,
143,
Mudd, S.H., Finkelstein, 240, 4382-4392.
J.D.,
Irreverre,
F. and Laster,
L.
(1965)
J. biol.
Chem.
Nagabhushanam, Nakagawa, Porter, 364,
H. and Kimura, P.N., Grishaver, 128-139.
Vesterberg, Warburg,
A. and Greenberg, H. (1968) M.S.
0. and Svensson, 0. and Christian,
D.M.
(1965)
Biochim.
and Jones, H. (1966)
W. (1941)
J. biol.
Chem. 240,
Biophys. O.W.
Acta, Biochem.
555
Res.
(1974) them.
Commun. 32,
Biochim. stand.
Z. 3&,
3002-3008.
0,
384-421.
Biophys. 820-834.
208-214. Acta,
