The Relation between the Functions Arabinofuranosyladenine as ...

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,