DIFFERENT

PATTERNS

OF

INHIBITION

DOPAMINE-;3-HYDROXYLASE Hiroshi

IZUMI,

BY

Hideko OYAMA, and Hikaru

Makoto

OF

CYSTEINE HAYAKARI

OZAWA

Pharmaceutical Institute, Tohoka University, Aobayama, Sendai 980, Japan Accepted December 18, 1975

SHORT by sulfhydryl hibition

compounds

COMMUNICATIONS

such as cysteine,

of DBH by these sulfhydryl

type reacting purified found

reagents

dopamine-f3-hydroxylase

(PDBH)

activity was dose dependent.

preparation

DBH activity

to the procedure

1.0 ml: potassium acid 10 /moles, The reaction the addition minated formed

by adding periodate,

at 37'C

to the procedure

et al. (3).

Reaction

for 5 min.

of norsynephrine

DBH activity was progressively

of norsynephrine

contained

in

fumaric

The reaction

by

was ter

of norsynephrine

by measuring

the absorbance

under these conditions

to the amount

of enzyme mixture

It can be concluded

by increasing

was started

to p-hydroxybenzaldehyde

of this product

from tyramine

by Fig. IA.

reduced

of NEM, whereas these inhibitions

mixture

acid 10 pmoles,

The amounts

of cysteine in enzymic reaction

of NEM is illustrated

chromatography to norsynephrine

The incubation

hydroxide.

by the conversion

The effect of the inclusion

of Foldes et al.

of tyramine

and carried out for 15 min at 37 °C in air.

lineally for 20 min and was also proportional

the presence

of DBH

10 ,'imoles, catalase 200 Sigma unit, and the enzyme.

followed by determination

at 330 mlq. The formation

we

or by

effect of cysteine

at the stage of DEAE-cellulose

2 nil of 4 N ammonium

were determined

cysteine,

of NEM

the extent of the inhibition

buffer (pH 5.5) 100 ,nmoles, ascorbic

was preincubated

of substrate,

with

by the addition

was assayed by the conversion

tyramine hydrochloride

by adding

when the partially

was pretreated

glands according

of Van der Schoot

phosphate

mixture

However,

of sulfhydryl

medulla of bovines.

In these studies the enzyme obtained

according

although

A and that the in

by the addition

In this note, we wish to report the inhibitory

from the adrenal

elute was used.

coenzyme

(NEM).

was not reversed

DBH was purified from beef adrenal (2).

and

was restored

preparation

of DBH activity

a dialysis of the cysteine-PDBH

on PDBH

glutathione

compounds

such as N-ethylmaleimide

that the inhibition

Japan. J. Pharmacol. 26, 264 (1976)

used. in the absence

and

from these data that the

cysteine concentration

were reversed by the addition

preceeded

of equimolar

in the absence concentration

( o. 1. (A): Effect of N-ethylmaleimide (NEM) on cysteinc inhibition of dopamine ,-hydroxylase. Samples were preincubated for 5 min and incubated for 15 min. DBH activities were assayed in the absence and the presence of NEM at a con centration equivalent to the concentration of cysteinc. (B): Effect of N-ethylmaleimide (NEM) on cysteine-pretreated dopamine-,% hydroxylase. PDBH preparation was pretreated with cysteine of various con centrations for 10 min at 37 C. An aliquot was then measured for DBH activities in the absence and the presence of' NEM at a concentration equivalent to the concentration of cysteinc. The term pCYST1::1NE is defined as negative logarithm of molar concentration of cvsteine.

SHORT of NEM.

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These results are in agreement

with previous

ever, when the PDBH

preparation

aliquot

of the mixture

was assayed

NEM,

the restoration

of the cysteine-induced

addition

of equimolar

NEM affected different

DBH

with cysteine for 10 min at 37'C,

and an

in the absence and the presence

inhibition

of DBH

of DBH was not observed

in different

for the inhibitory

the possibility cysteine

with cysteine for 10 min at 37-C,

et al. (1). How

of

by the

of NEM (Fig. I B). As can be seen in Figs. IA and 1B,

are responsible

by removing

findings by Nagatsu

for enzymic activity

concentrations

We also investigated treatment

was pretreated

the cysteine inhibition

mechanisms

Japan. J. Pharmacol. 26, 265 (1976)

ways.

effect of cysteine.

of reversibility

by dialysis.

These results suggest that

of the inhibition

The PDBH

then dialyzed against

of cysteine-pre

preparation

200 volumes

was pretreated

of 0.05 M phosphate

buffer, pH 6.5 at 4 'C for 24 hr. The buffer was changed three times under dialysis, and then, an aliquot

of the mixture

range of 1.25-10 mM.

was assayed

for DBH

activity

in the presence

of substrate

in the

The results are shown in Fig. 2. As can be seen, the decrease in DBH

activity did not recover

by dialysis.

Since the observed

is unaltered

by the cysteine pretreatment

responsible

for the inhibition

of DBH

Km value (0.83 mM) for substrate

(Fig. 2, inset), it seems likely that the mechanism activity

by cysteine

pretreatment

is not associated

Cu has been suggested

for several sulfur

with the decrease of the affinity of the enzyme for substrate. Inhibition containing

of DBH by binding of the enzyme

compounds:

diethyldithiocarbamate

(5, 6), dimethyldithiocarbamate

(7), D-cysteine,

A (1). As shown above, the cysteine-pretreated addition

of NEM

addition

of Cu ++ (data

and

disulfiram

L-cysteine, inhibition

(Fig. I B), by dialysis of the cysteine-PDBH not shown),

suggesting

DBH by cysteine would not be through data and previously

reported

observations

that

the binding

(4),

thiourea

mercaptoethanol

and coenzyme

of DBH was not reversed preparation

the mechanism

by the

(Fig. 2) or by the

of this inhibition

of the Cu of DBH.

(1, 8), it might

derivatives

be reasonable

of

From the present to consider

Ftn. 2. Effect of dialysis of cysteine-pretreated dopamine- ,-~-hydroxylase on kinetic parameter. PDBH preparation was pretreated with cysteine for 10 min at 37 "C, and, after dialyzed the mixture, DBH activities were assayed in the presence of various concentrations of tyramine (final cysteine concentration, 10_1 M). Line Weaver-Bark plots (inset) of tyramine concentration against rate of hydroxylation with native (-) and cysteine-pretreated (-s-) enzymes. The velocity is expressed as /moles of norsynephrine formed per 15 min.

that

SHORT the inactivation

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Japan. J. Pharmacol. 26, 266 (1976)

of DBH by cysteine in the absence of catalase

(H202) rather than cysteine itself.

is due to hydrogen

The result in Fig. 2 also suggests that modification

maximum

velocity to tyramine may be simply due to the destruction

However,

precise mechanism

of the inhibition

In the presence of the naturally enzyme

of biosynthesis

such inhibitors laboratories

occurring

of norepinephrine

from various

(2, 10-15).

(13, 14), we observed

naturally

occurring

inhibitors

finding and of wide distribution that

and characterization

species have been investigated the properties

that the mechanism to that

of the naturally of inhition

of the cysteine

groups in the molecule.

of the naturally

these inhibitors

DBH can become the rate-limiting

The purification

seems to be similar

seem to have sulfhydryl

of the

of a part of active DBH.

unknown.

inhibitors, (9).

tissues of different

of DBH

14), it was suggested

remains

In the process of studying

inhibitors

that these inhibitors

peroxide

occurring

might inhibit

inhibitors

of

in various occurring

of DBH inhibition,

by the and

In view of the present in animal

the DBH irreversibly

tissues (13, in 1417oand

play an important role in the regulation of norepinephrine synthesis. Further investigation is underway to determine the mechanism of the irreversible inhibition of DBH by cysteine pretreatment. REFERENCES I) NAGATSU,T., KUZUYA,H. AND 1-IIDAKA,H.: Biochim. Biophys. Acta, 139, 319 (1967); 2) FOLDFS,A., JEFFEREY,P.L., PRESTON,B.N. AND AUSTIN, L.: Blochem. J., 126, 1209 (1972): 3) VAN DER SCHOOT,J.B., CREVFLING,C.R., NAGATSU,T. AND UDENFRIEND,S.: J. Pharmacol. exp. Ther., 141, 74 (1963); 4) GOLDSTEIN,M., LAUBER,E. AND MAKEREGHAN,M.R.: J. biol. Chem., 240, 2066 (1965); 5) JoHNsoN, G.A., BoUKMA,S.J. AND KIM, E.G.: J. Pharmacol. exp. Ther., 168, 229 (1969): 6) OYAMA, H., IzuM1, H. AND OZAWA, H.: Biochem. Pharmacol., 25, 277 (1976); 7) LIPPMAN,N. AND LLOYD,K.: Blochem. Pharmacol., 18, 2507 (1969); 8) KUZUYA, H.: J. lit-ate med. Ass., 19, 401 (1967); 9) CREVELING,C.R., DALY, J., WITKOP, B. AND UDEN FRIEND,S.: Biochim. Biophys. Acta, 64, 125 (1962); 10) AUSTIN, L., LIVETT,B.G. AND CHUBB, I.W.: Circulation Res., Suppl., 10(111) Ill (1967): 11) DUCH, D.S., VIVEROS,O.H. ANDKINERRSH N.: Blochem. Pharmacol., 17, 255 (1968): 12) DUCH, D.S. ANDKIRSHNER,N.: Biochem. Biophys. Acta, 236, 628 (1971): 13) OYAMA,H., IzuM1, H. AND OZAWA, H.: Yakugaku Zasshi 95, 621 (1975) (ill Japanese); 14) IZUMI, H., OYAMA,H. AND OZAWA, H.: Cheer. Pharm. Bull., Tokyo, 23, 2362 (1975): 15) HARRALSON,J.D. AND BROWN,F.C.: Proc. Soc. exp. Biol. Med., 149, 643 (1975)

Different patterns of inhibition of dopamine-beta-hydroxylase by cysteine.

DIFFERENT PATTERNS OF INHIBITION DOPAMINE-;3-HYDROXYLASE Hiroshi IZUMI, BY Hideko OYAMA, and Hikaru Makoto OF CYSTEINE HAYAKARI OZAWA Phar...
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