Jun 22, 1981 - S-[5-(9-arabinofuranosyladenyl)}-L-homocysteine;. Ado, .... Cory et al.,. 1965). These inhibitors potentiate the oncostatic effects of ara-.
0022-3565/81/2183.075402.00/0
THE JOURNAL OF PHARMACOLOGY Copyright JO 1981 by The American
AND EXPERIMENTAL Society for Pharmacology
THERAPEUTICS and Experimental
The Relation between Arabinofuranosyladenine S-Adenosylhomocysteine S. HELLAND
and
Accepted
for
publication
University
June
22,
of Bergen
School
of Medicine,
P. M. Ueland:
The
relation between as inactivator hydrolase. J.
was
further
and
by
to adehydrolase
with
(ara-A) was converted nine in the presence of S-adenosylhomocysteine (EC 3.3.1 .1 .) and to adenine and S-[5’-(9-arabinofuranosylad(ara-AHcy)
when
L-homocysteine.
The
the
incubation
formation
of
mixture
adenine
pro-
ceeded until 3.3 mol of adenine was formed per mol of enzyme, and at this point the enzyme was totally inactivated. In the presence of homocysteine, the rate of ara-AHcy formation was about half the rate of adenine formation. The association of the conversion of ara-A to adenine with the inactivation process
Investigation
of the
the antibiotic development
ara-A of
biochemical
(Cass,
potent
(Woo
3-nonyl)-adenine
(Schaeffer
the metabolic ara-Hx (Brink 1965).
inhibitors
These
LePage The
cells
et al., to
reductase
ara-ATP,
(Moore
Schwender,
to the
inactive
Cohen,
1966;
the
al.,
1975)
et al.,
Cohen,
oncostatic
1967)
Cohen,
1967,
1968;
LePage,
ration
into
nascent
DNA
may
inhibit
metabolite Cory
of ara-
et
1978)
1977;
a!.,
1976).
attributed
and
replication
its
(Tsang
to its
of
is
a
potent
from
mocysteine; 758
ara-A,
9-fl-D-arabinofuranosyladenine;
ara-Hx,
of these
presence
NAD.
A
is
an
ATP
of
processes
homocysteine
of adenine were to the synthewas associated
Adenine
or the
sub-
1979; both
inhibitors
but
congener,
are
from the
increasing some
studies
finding
mouse
far
that
potent
effects a product
this
in
en1979;
elevates
Ueland,
respect
than
their
These
level
in of
et al., in favor
ob-
agreement AdoHcy
in
Thus,
possibility
degradation of 5-adenosyl-
reactions
proposed
data
1980).
of the
blocking inhibitr
transmethylation
araliver
are
the
is accumulating
1981; and
mouse
enzymes
(Zimmermann
as originally
liver
this
the
1981).
of ara-A stem from from and a potent
1980)
and of
Ueland,
isolated
ara-A
rat
ara-AMP
1979).
inactivators
and
lymphocytes
evidence
Chiang,
less
with
methionine-dependent and
and
(Helland
ara-A
from
inactivates (Hershfield,
(Helland al.,
et
of
nucleoside
liver (Helland and Ueland, process suggest that ara-
agent
Hershfield
this
hydrolase
and mouse inactivation
site-directed
active
enzyme,
of the
mode that
irreversibly and placenta
lymphocytes 1979)
findings
AdoHcy
and
1976)
human
are
by the
of
Kerr,
et a!.,
intact
March 20, 1981. by grants from Nordisk Insulinfond, Norwegian Norwegian Research Council for Science and the Fond.
AdoHcy, S-adenosylhomocysteine; ADA, adenosine deaminase; Hepes,
inhibitor and
AdoHcy,
5’-triphosphate;
6 Haukeland
A nucleotide-independent
is suggested
1981). The kinetics
with
DNA
kinetics
in the
bound
1980).
et a!.,
Lee
tamed
incorpo-
of enzyme
of ara-A
action
Hershfield,
Au-Yeung,
that
of the enzyme and formation i.e. , by a factor corresponding Inactivation of the enzyme
reduction
that
ABBREVIATIONS:
N-501
stance liberating adenine was tightly bound to the enzyme, whereas ara-AHcy was dissociable. These data suggest that inactivation, adenine formation and reduction of NAD result from an abortive catalytic cycle, whereas enzyme entering a complete catalytic cycle leading to formation of ara-AHcy is not inactivated. The fact that the inactivation and the enzyme catalysis occur simultaneously at about equal rates offers an opportunity to demonstrate the competing relationship between these two processes.
Hershfield
al.,
et
by the
observation
both inactivation reduced by 30%, sis of ara-AHcy.
zyme
of ribonucleotide DNA polymerases
and
the
inhibiting
et al.,
Schabel
been
is an inhibitor
and
and (Lee
vito
1979;
have
(Furth
Received for publication I This work was supported Society for Fighting Cancer, Humanities and Langfeldt.s
of Dermatology,
demonstrated
the
(Trewyn
effects
Cass
1977; and in
of ara-A which
and
1974),
1964a,b;
et
of
by the like 2’-
erythro-9-(2-hydroxy-
of ara-A
Plunkett
effects
properties
stimulated inhibitors
and
potentiate
1976;
biological been
deaminase 1974)
and
(Borondy and Cohen,
biological
conversion
et al.,
conversion and LePage,
A in intact 1976; Plunkett
and has
1979)
adenosine
deoxycoformycin
and the Department
the funcand subPharmacol.
9-,f3-D-Arabinofuranosyladenine
enyl)]-L-homocysteine
MFH-bygget
1981
tions of 9-f3-o -arabinofuranosyladenine strate of S-adenosylhomocysteine Exp. Ther. 218: 758-763, 1981.
contained
of
by Trewyn
of
(Cantoni and
Kerr
(1976).
9-/JD-arabinofuranosylhypoxanthine;
ara-AHcy, S-[5-(9-arabinofuranosyladenyl)}-L-homocysteine; N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic
ara-ATP,
9-fl-D-arabinofuranosyladenine Ado,
acid.
adenosine;
Hcy,
DL-hO-
Downloaded from jpet.aspetjournals.org at Swets Blackwell IncUniv i Bergen on May 26, 2009
ABSTRACT Helland S. and
the Functions of 9-fl-Das Inactivator and Substrate Hydrolase1
P. M. UELAND
of Pharmacology, Bergen, Norway
Department
sykehus,
VoL 218, No. 3 Printed in U.S.A.
Therapeutics
1981
ara-A
Our
present
knowledge
anism of action lism of AdoHcy, The the
of the
of
with
of ara-A
of the
present
of ara-A,
interference in figure
ara-A
conversions
subject
metabolism
its possible summarized
interaction
metabolic is
and are
with
in the
(Rf
main
and
from
an
abortive
homocysteine,
rate
catalytic
comparable
enzyme 3) The
to
that
the
of
a complete and the
entering inactivation
cycle.
of the
In
from
presence
of
proceeds
process,
at a and
the
cycle is not inactivated. catalysis are competing
Chemicals.
Ado,
from
I
purchased
calf
from
kindly
Chemical
Sigma
provided
Chemotherapy,
by
20
(1967). the
cm)
were
Cancer
prepared
was
Centre,
(0.25
used.
Isocratic
(23#{176}C).The
elution a
and
Hcy
(3
fraction
and
were
developed
coupled
thin-layer
the
Polyethylplates
and
Randerath
was
obtained
UV
(20
at ambient
elsewhere
(Ueland
[‘H]ara-A
mm
37#{176}C in
30
from
compound
as follows. at
mouse
liver.
was
synthe-
(200 the
The
tM)
and
presence
of
incubation
buffer
were
subjected
to high-pressure
liquid
chromo-
were in
in series
after
the
a
[:IH]araAHcy
for
AdoHcy
synthase
method
The
incubation
mM)
in buffer
mixture A. The
for
the
(in buffer
A).
The
indicated.
into
performed (0.25
some
UV
on polyethylThe chro(2:1:1). Ade-
separate mM
reaction
was
Hepes
and
of
terminated
mM
(100
zM)
and
cell
one
by
the
dithiothreitol
B. The and ara-A addition at The
recorded
after
The
by adding
scintillation
(1.3
hydrolase with Hcy
samples
mg/nil)
of 25
and the previin the
counting. and
are-A
(100
M)
in
5 mM
330
to nm
the
enzyme.
spectrum
increase
in
Optical
a Cary
using
difference
the
(3
era-Alley.
acid
by liquid
of are-A
recorded
Hcy
magnesium acetate, 150 mM (buffer B) were placed in one cell of (light path, 0.493 cm X 2). The other cell reference cuvette contained enzyme in buffer in buffer B in the other. The reaction was
cuvette
buffer
acid.
Enzyme containing
7.0)
of protein.
Determination
ADA inhibitors
Ado
model
between
absorbance
had
density
was
219 recording 450 and 280 nm
nearly
come
to
a
halt.
DNA
.‘
was
determined
(pH
2
contained
was
a final
37#{176}C.
of ara-A to aden.ine and the presence of AdoHcy mixture was supplemented
incubation
The
spots
B in started
was
was neutralized subjected to thin-layer chromatography as described (Ueland and Sab#{248},1979a). The radioactivity residing
ously
KC1
to
was performed by a and Sb#{248},1979a).
(Ueland
enzyme,
30 zl of 0.8 N perchloric
solution
15
water
37#{176}Cin
at
Spectrophotometry.
mm).
in isobutanol-ethanol-water
contained
conversion
incubated
This
elsewhere
temperature
was
when
in distilled
activity.
described
[3H]ara-A
l
resuspended
was
of 51 tM.
Assay
temper-
and
and
Assay from
detector
on
radiochemical
Inhibition
f
for
(4 mg/nil)
spectrophotometer.
dCF
mouse liver. The ena slight modification
published
This
[:iH]aA
supernatants
continuously
EHNAJ
ara-AHcy
15 mM Hepes (pH 7.0) containing 0.2% bovine serum albumin, 150 mM KC1, 5 mM magnesium acetate and 2 mM clithiothreitol (buffer A). The reaction was stopped by heating for 5 mm at 100#{176}C and the denatured protein was removed by centrifugation. Samples of 100d
ara-A
glass
recorded
sheets
by
procedure
incubated
a two-compartment
was
from
homogeneity
and
0.35)
was
concentration
collected.
This
cellulose
out
were
hydrolase
iized
Program,
MD. on
8300
was
nm
was
ml were
chromatography.
eneimine-impregnated
carried
at 254
collector
of 0.66
Thin-layer
was
SP
=
1977). from
mM)
were
England.
a model
(2 ml/min)
absorbance
Fractions
matograms
mm)
by Randerath (22 Ci/mol)
Amersham,
chromatograph
ature monitor.
MO
Bethesda,
sheets
(R
of [2,8-3H]ara-AHcy.
enzymatically
ADA Hepes
liquid chromatography. Samples were deproteinto 100#{176}C or by mixing with perchloric acid followed by as described elsewhere (Ueland and Sb#{248},1979c). Preremoved by centrifugation. A Spectra Physics model SP
liquid
experiments
and
Therapeutics
described
as
ADP
St. Louis,
Institute,
ara-Hx
Ci8-tBondapak column using 8% methanol in water as solvent. The fractions corresponding to a UV absorbing peak eluting at a higher retention time than ara-A and adenine, and which cochromatographed with radioactive material, were collected, pooled, lyoph-
(DL-homocysteine),
Company,
[2,8-1H]Adenine-$-D-arabinoside
High-pressure ized by heating neutralization, cipitate
Hcy AMP,
Developmental
thin-layer
Radiochemical
8000B
the
National
eneimine-impregnated
x
adenme, mucosa),
apparent
of a purification
Preparation sized
from
and Methods
AdoHcy, intestinal
to
D#{248}skeland,
AdoHcy
0.52),
=
759
Hydrolase
the
absorbance
(E
nm)
at
of 13.0
for
280 the
Protein
nm
using
was a
(Ueland
enzyme
determined
specific
by measuring
extinction
et a!.
coefficient
1978).
synthesis
Results
a r a-H x’a
ra- A
-
-
-
a ra -A I P
Formation [3H]araA
of incubated
converted
to
(fig.
the incubation an additional
Ado
AdoHcy
:-
Hcy
hydrotose
AdoMet
was
1 . Metabolism hydroxy-3-nonyl)adenine; osylmethionine.
and
mode of action of ara-A. dCF, 2’-deoxycoformycin;
gram. The formation [3H]ara-A was dependent
of
identified
and heating
as
EHNA, erythro-9-(2S-aden-
the
plates.
radioactive
Based
on
compound
adenine
and
enzyme
concentration
ara-AHcy
these was
formed (data
was
with
detected
was
by When
homocysteine,
in the
data,
not
observed
when
incubation
it was
[3H]ara-AHcy. shown).
chromato-
compound from of ara-A, homocys-
before
increased not
adenine
chromatography.
this radioactive on the presence
2) by
from era-A. hydrolase, was
the not
(data
shown). The isolated compound was a substrate hydrolase in the hydrolytic direction (see below) matographed with AdoHcy on polyethyleneimine
mcn
AdoMet,
was
supplemented
peak
(fig.
adenine of AdoHcy
thin-layer
mixture radioactive
thin-layer Fig.
which
2) and
teine and enzyme enzyme was denatured
{ltion
and presence
a substance
reverse-phase
ADA
ara-AHcy in the
for AdoHcy and cochrocellulose concluded The
as a function
that
amount
of
of the
Downloaded from jpet.aspetjournals.org at Swets Blackwell IncUniv i Bergen on May 26, 2009
Materials
was
1978)
(R
AdoHcy
senarated in this system. of AdoHcy hydrolase
purified
tography
processes.
(type
was
and
derived
the
ara-A
inactivation
catalytic enzyme
ara-A
0.61),
were
(Ueland,
enzyme,
conclusions
2)
of ara-AHcy
synthesis
=
0.15)
zyme
the
from data in this report are as follows. 1) Inactivation of the enzyme by ara-A is associated with conversion of ara-A to adenine and reduction of enzyme-bound NAD. These events result
(Rf Purification
hydrolase
presence
The
nine
metabo-
1.
AdoHcy
work.
its mech-
the
and
760
Helland
and
Vol. 218
Ueland and
enzyme
formation
corresponding
5)
uE
to the
Reduction
CC 0 -:
of
of rate
adenine
was
of formation
reduced
by
a factor
of ara-AHcy.
enzyme-bound
NAD.
The
reduction
of
enzyme-bound NAD during the inactivation of the enzyme ara-A was followed by recording the increase in the absorbance at 330 nm (Abeles et at., 1980). The change in absorbance
(A
.0
followed obtained
the inactivation of the enzyme by at 20#{176}C is shown in figure 5. The
obtained
at
change
the
in
recording
precipitate after
40
shoulder
C 0
37#{176}C,except
absorbance
of after
that
was
absorbance prolonged
mm of incubation at 320 to 340 nm
the
higher.
At
rate
of
high
temperature
was
obscured
incubation.
The
at and
ara-A. same
The result result was
and
inactivation
(37#{176}C),
by
20#{176}Cwas a maximum
by
formation
of
difference
spectrum
characterized at 300 nm
(data
by
a not
shown).
Separation
U
E
tion. Samples and AdoHcy
0.
matography
U
were
‘C
analyzed
of free
and
bound
metabolites
by
from a incubation mixture containing hydrolase were subjected to Sephadex 30 mm
after by
of incubation.
thin-layer
The
chromatography.
gel
filtra-
[3H]ara-A G-25 chro-
separate fractions The elution pro-
0
10 Retention Fig. 2.
15 (mm)
time
and its metabolites by reverse-phase liquid (5 j.M) was incubated for 30 mm in the presence of AdoHcy hydrolase (1 1 3 g/ml) (B), enzyme and 3 mM Hcy (C) or Hcy (3 mM) without enzyme (D). Deproteinized samples (50 jil) from the incubation mixture were analyzed by high-pressure liquid chromagraphy on a C8 Bondapak column using 1 0 mM acetate buffer (pH 5.5) containing 8% methanol as solvent. Fractions of the effluent were collected and radioactivity was determined by liquid scintillation Separation
of ara-A
chromatography.
counting. Time nine.
[3H]ara-A
A shows
the elution
course
for
The
which
condition
(Helland
adenine
ratio
for
and
paralleled
(fig.
ara-AHcy
(MW
Relation
=
In the
185,000)
between
of the enzyme lase by ara-A reduced
was
by
The
formed
formation
presence
2
Deviation
of
from
homocysteine,
linearity
may
rate
Kinetic
the
rate
be explained
bs
The
of enzyme inactivation
of AdoHcy hydrofor the first two half-
data for adenine formation showed that the A to adenine closely followed the inactivation the absence and presence of homocysteine the
the
constant.
and
the 4A).
and after the
of ara-A
was 3.3 mol/mol et a!., 1978).
The inactivation order kinetics (fig.
ade-
in that
remained
of homocysteine,
one-third
conversion
of ara-AHcy
2:1 and
adenine
by ara-A. showed first presence
1981).
(Ueland
and
of ara-AHcy linear phase elapsed before
of ara-A (figs. 3, A and of the enzyme under this
formation
of adenine
amount
3)
lives.2
of
Ueland, the
[adeninej/[ara-AHcy]
maximal
was
formation
off at high concentration with rapid inactivation
curves levelled B), consistent to
the curves
of standards.
the formation were characterized by an initial a plateau was observed. Short time
adenine
progress
profiles
of
of
conversion
of
process (fig.
4B).
both Thus,
inactivation
consumption
15
of inactivation treatment
of
of ara-A.
Time
the
Fig.
ara-
in in the
3. Progress
curves
30 of
incubation
for the formation
4 (mm)
of adenine
and
ara-AHcy.
The time course of the formation of adenine (A) and ara-AHcy (B) from ara-A in the presence of AdoHcy hydrolase (200 ,zg/ml) was determined at an initial concentration of 1 zM (, #{149}), 3 tM (V, V), 1 0 jzM (0, ), 30 tM (Lx, A) and 1 00 jiM (0, #{149}) of ara-A. The incubation mixture contained 3 mM Hcy. Panel C shows the ratio obtained when dividing the concentration of adenine by the concentrations of araAHcy. The same symbols are used as in panel A.
Downloaded from jpet.aspetjournals.org at Swets Blackwell IncUniv i Bergen on May 26, 2009
0
1981
are-A concentration C
pound
5) U 0 0.
radioactivity
small
fraction
0C
E
adenine,
C
was
mainly
whereas in the
only
and
as
the
same
(data
not
shown).
only
stable
in the
Thus,
this
of ara-A
a
for at
conditions
by converting
of ADA)
com-
ADA.
and
was
under
amount
the
excess
adenine
7). [:;H]araAHcy
small
presence
when
hydrolase
recovered
(fig.
761
Hydrolase
observed
AdoHcy
ara-Hx
as
AdoHcy was
with
mm when incubated of AdoHcy hydrolase acts upon ara-AHcy
ara-Hx
5)
[3H]ara-AHcy
incubated
The
least 60 absence hydrolase
a)
of
was
and
AdoHcy
compound
to
(deaminated
to
is formed.
Discussion
>
U
Inactivation
Ueland, of
the
and
AdoHcy
hydrolase
Hershfield,
nucleoside
1979)
to
conversion
the
enzyme
of ara-A
(fig.
1).
The
by
ara-A
is associated (Helland
to
adenine
formation
of
(Helland
with and
binding
Ueland,
1981)
or a substance adenine
and
tight
liberating
seems
to
be
closely
0
linked
‘C
to the
nine C
0
inactivation
formed
process of enzyme
per mole
content
of the
suggest
a similar
enzyme
(Palmer
‘C
OC
.E
in position
3 of
the
dized
by enzyme neously liberates
C C
NAD
a)
‘D
(fig.
5).
drolase
This
ara-A
mechanism from Ado
10
Time
Fig. 4. Kinetics
of inactivation
adenine in the absence lase (1 00 tg/ml) was and presence
of
incubation
(mm)
of AdoHcy
and presence preincubated
hydrolase
and formation
of homocysteine. with
of 3 mM Hcy in bufter
AdoHcy
5 tM
[3H]ara-A
in the
A. After
various
periods
of
hydroabsence
of time,
aliquots (1 0 tl) were added to perchloric acid, neutralized and the amount of adenine formed determined by thin-layer chromatography. From the same preincubation mixtures, samples (5 sl) were transferred to the incubation mixtures (300 gil) containing 1 00 sl of [‘4C]Ado and 3
mM Hcy and assayed for AdoHcy synthase activity. The incubation time was 2 and 6 mm. Panel A shows a semilogarithmic plot for the inactivation of the enzyme in the absence (#{149}) and presence (0) of homocysteine. Panel B shows a semilogarithmic plot for the formation of adenine
under
the
same
to adenine has in the
that
placenta and
of
the
These
loss
data for
the
group is
oxi-
spontareduction of
with
inactivation
the
findings
of AdoHcy with
is associated of enzyme-bound
hy-
conversion
NAD.
been proposed for the presence of the mouse
hydro-
nucleoside
is in accordance
showing
human
1979).
of AdoHcy et a!. (1980) The hydroxyl
NAD. The 3’-keto-nucleoside leading to a irreversible
suggestion
(1980)
from
Abeles,
furanosyl-residue
bound adenine
of Hershfield
LJ
and
of inactivation by Abeles by 2-deoxy-Ado.
mechanism
lase by ara-A as proposed inactivation ofthe enzyme
4 LJra;1
(fig. 4). The amount of adeinactivated equals the NAD
of
A similar
formation of adenine liver enzyme (Ueland
and Helland, 1980). In the presence of homocysteine,
AdoHcy
the formation of ara-AHcy formation of adenine plus
ara-A (fig. 2). The rate of in the presence of homo-
cysteine
equals
the
rate
of homocysteine.
from ara-AHcy
of formation
of adenine
both
Furthermore,
hydrolase
the
velocity
catalyzes
in
the of
the
absence
macti-
-
conditions. 0.
files
for
portion
ara-A
of adenine
adenine was
and
was
adenine was
mainly
liberated
from
are
eluted
shown in the
void
protein-bound the
in figure volume,
and
enzyme.
Only
6A.
only a
The
major
showing
that
a small
small
fraction
portion
of
the
0)
C
o0
0
3
C
00
E
‘C >(
radioactivity A. Exactly
associated the same
with results
the enzyme were obtained
was
identified when the
as arafractions
0
>
0 >
were deproteinized “Materials and
with Methods”)
before chromatographic In a similar experiment, containing
filtration. are
shown
[3H]ara-A, The elution in figure
in the protein-bound in the free fraction. complex completely Hydrolysis
perchloric or by
acid heating
(as described (100#{176}Cfor
analysis (data not shown). samples of an incubation Hcy and enzyme profiles for adenine,
6B.
The
fraction, These
is characterized dissociated ofara-AHcy.
main
by sufficiently before
or during
of adenine
appeared
ara-AHcy was recovered that ara-AHcy-enzyme low
half-life
to be nearly
chromatography.
A time-dependent
U
4
mixture
were subjected to gel ara-A and ara-AHcy
portion
whereas data show
under 5 mm)
decrease
10 Time
Fig.
5. Relation
and
increase
incubation
inactivation
30 (mm
of AdoHcy
hydrolase
by ara-A
in absorbance at 330 nM. Inactivation of the enzyme in the presence of 1 00 tM ara-A at 20#{176}Cwas determined as described in legend of figure 4. In a parallel experiment, the increase in absorbance at 330 mg/mi) by
difference
in the
between
20 of
to 1 -cm
nm was 1 00 zM between
light-path)
recorded during inactivation of the enzyme (1 ,3 ara-A at the same temperature. 00 is the the maximal absorbance (ODmax 0,1 5 adjusted and the absorbance at each time point (OD,).
Downloaded from jpet.aspetjournals.org at Swets Blackwell IncUniv i Bergen on May 26, 2009
adenine
0
of
1981;
762
Helland
and
Vol. 218
Ueland AdoHcy
hydrolase
thesis
of
agreement AdoHcy
U ‘C
0
0
number
Thus,
determination
of catalytic
the the
C
cycle
and
leading
catalyzes
the
homocysteine
to
syn-
is not
that ara-A is not a substrate et al., 1981; Zimmermann is a poor in quantities
sites of
catalytic
hydrolase
ara-A
However, ara-AHcy and is not formed
of the
usually
from
with the statement hydrolase (Guranowski
tration of enzyme the concentration 5)
enzyme. that AdoHcy
ara-AHcy
a!., 1980). hydrolase
E a
an abortive
enters
inactivation of the The observation
on
the
ara-AHcy
substrate exceeding enzyme
(see
formation
which is of the of its substrate(s),
for
AdoHcy one-third
“Results”).
requires
same order whereas
in of et
concen-
of magnitude as enzyme activities
are measured with substrate in excess. presence of Ado, AdoHcy and other adenine intact cell may inhibit the formation ofara-AHcy.
Furthermore, derivates in This may
C
explain
0
cell-free
that
ara-AHcy
system
lymphoblasts
0
is not
(Guranowski (Zimmermann
formed
in detectable
et a!., 1981) et a!., 1980).
or
in
amounts
in intact
mouse
C 0
>
U I 0
a
. 4 0
0
Fraction
Fig.
6. Separation
number
of free and bound
0
metabolites
by Sephadex G-25 (1 zM) was incubated at 37#{176}Cwith in buffer A. After 30 mm of incubation, to chromatography on a Sephadex G1 5 mM Hepes containing 1 0 mM 2-merthe same buffer. Fractions of 1 44 sl were perchloric acid, neutralized and analyzed The elution profile for ara-A and adenine B shows the results of a similar experiin this experiment was supplemented profiles for ara-A, adenine and ara-AHcy
chromatography. A, [3H]ara-A AdoHcy hydrolase (30 sg/ml) a sample of 50 tl was subjected 25 column equilibrated with captoethanol and eluted with collected, deproteinized with by thin-layer chromatography. is shown on the figure. Panel ment. The incubation mixture with 3 mM Hcy. The elution are shown.
Fig. 7. Hydrolysis in the
buffer
third
process when
mocysteine reaction
and
formation
the
incubation
(fig.
4). These
scheme
is converted
to
observations the
can
be
8. Formation
with
enzyme
at
hydrolase, This
ara-A.E NAD
by
to the
a rate
a
parameters complete
only
adenine
small
amount
suggests
that
the
of ara-A ara-AHcy
U/mI)
in
ne.E
NADH
Hcy k2Jt
a ara-AHcy.E
is
NAD
complex
corresponding
rate is: k catalytic
in
(50
3’-keto-ara-A.E +
to ara-AHcy
constant,
