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.
COMMUNICATIONS
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
COMMUNICATIONS
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)