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Some properties of tyrosine hydroxylase from the caudate nucleus
Life Sciences Vol . 3, pp . 1195-1200, 1964. Pergamon Press, Inc . Printed in the United States .
SOME PROPERTIES OF TYROSINE HYOROXYLASE FROM THE CAUDATE NUCLEUS S . P . Bagchi and P .
L . McGeer
Kinsmen Laboratory of Neurological Research, The University of British Columbia, Vancouver, Canada
(Received 4 September 1964)
The least understood step
in the biosynthesis of the catecholamines is
the conversion of tyrosine to 3,4-dihydroxyphenylalanine (Dopa) . investigators have demonstrated, takes place, Nagatsu et al . tions .
Although many
both in vivo and in vitro , that this conversion
(1) were the first to do so using cell-free prepara-
They described some of the properties of tyrosine hydroxylase obtained
from brain stem and other organs . In brain, the caudate nucleus has the highest concentration of dopamine . Moreover,
it has been found to be quite active in converting tyrosine to catech-
olamines both
in vivo (2) and
in vitro (3) .
This report deals with the partial
purification of tyrosine hydroxylase from beef and rat caudate nucleus, some properties of the enzyme, and the effect of several compounds on
its activity .
Methods Beef brain, obtained from a slaughterhouse, was thoroughly chilled within twenty minutes of killing in
ice cold, physiological saline .
rapidly rgnoved from decapitated animals .
Rat brain was
Dissection of the caudates and aii
subsequent steps were performed at 0-S~C . Caudate tissue was homogenized
in ten volumes of
isotonic sucrose .
Whole
homogenate was either incubated at this stage, or subJected to centrifugation . 1195
1196
SOME PROPERTIES OF TYROSINE HYDROXYLASE
Vol . 3, No . 10
Cellular debris and nuclear material were removed by centrifuging at 600 G for ten minutes .
In the case
of
rat tissue, the supernatant was then centrifuged
at 20,000 G for fifteen minutes ;
in the case of beef tissue the supernatant was
centrifuged for a similar time at 12,000 G, pended
in
The sedimented fraction was resus-
its original volume of 0 .1 M phosphate buffer, pH
were treated by precipitation with
60'6
6.4 .
Beef particles
saturated ammonium sulfate .
The protein
precipitate was centrifuged off, and used as the enzyme source . Incubations were carried auf
in air at 37~C for one hour,
Each 25 ml
Erlenmeyer flask contained 270,000 cpm of L-tyrosine-U-C 14 ( .67 millimicromoles, specific activity
360
ing approximately
60
me/mM} ; 2,0 ml of 0 .1 M phosphate buffer, pH
6,4,
contain
mg of protein from the ammonium sulfate precipitation, or
alternatively the equivalent of 280 mg of wet tissue; and 0 .1 ml of an aqueous solution of ical
inhibitor or 0 .1 ml
of
water .
The blank in each case was an ident-
incubation except that the solution containing the enzyme was heated to
$O~C for ten minutes prior to incubation, The reaction was stopped by " adding 2 ml of 0 .8 N perchioric acid . solution was homogenized, centrifuged, and the catecholamines
The
in the supernatant
separated on an alumina column (4) . An aliquot of the column eluant was counted in a liquid scintillation spectrometer . parallel
The counts
in the test eluant minus those in the eluant from the
incubation with inactivated enzyme were taken as a measure of the rela
tive conversion of tyrosine to catechols by tyrosine hydroxylase . In a number of cases, a further aliquot of the eluant was subjected to paper chromatography to separate the products,
Approximately 20 micrograms of
cold carrier Dopa, dopamine, noradrenaline, dihydroxyphenylacetic acid and tyrosine were added and the mixture chromatographed in n-butanol 1N HC1 for
26
hours
(5) .
saturated with
The various areas were cut out and counted directly
in a liquid scintillation spectrometer containing 80-90% of the total
(6) .
Dopa-dopamine formed a single spot
activity on the strip,
The remainder was
tyrosine (6-10%), noradrenai3ne (4-5%} and dihydroxyphenylacetic ac td
in
(0-2°~) .
Vol . 3, No. 10
SOME PROPERTIES OF TYROSINE HYDROXYLASE
119 7
In cases where it was desired to separate Dopa and dopamine, a separate aliquot was chromatographed
in n-butanoi saturated with iN fiCi as before, the
area containing Dopa-dopamine eluted with an ethanol/ .03N HC1 mixture (3 :1), and the eluant respotted and run in methanol/n-butanoi/benzene/water (2 :1 :1 :1) . This system separates Dopa from dopamine (5) .
The areas containing these com-
pounds were cut from the paper and counted as before, Results and Discussion A preliminary experiment with rat caudate indicated that the major part of the enzyme activity could be located in the 20,000 G particulate fraction (Table 1),
A lower speed of centrifugation was employed for separating beef TABLE 1
Formation of Catecholamines from Tyrosine by Various Brain Fractions
Species
Preparation
Catecholamines formed per gm wet tissue per hr mN moles
Rat s`
Beef`
cpm
Whole homogenate
2 .23
10 .4x104
Supernatant
0 .06
0 .3x104
Particulate (20,000 G)
1 .69
7 .9x104
Whole homogenate
0 .97
10 .3x104
Supernatant
0 .33
3 .6x104
Particulate (12,000 G}
0 .63
6 .7x104
~ L-tyrosine used : 0 .25 pc at 3,Oxi0 -6 M final concentration ~ L-tyrosine used : 0,25 pc at 3,ßc10 - 7 M final concentration caudate fractions which might account for the higher activity in the supernatant using this tissue,
The ammonium sulfate precipitate from beef caudate showed a
two-fold increase in activity over the particulate fraction on a protein basis, The enzyme proved to be extr~nely unstable .
The activity of the ammonium
119 8
SOME PROPERTIES OF TYROSINE HYDROXYLASE
Vol . 3, No . 10
sulfate precipitate disappeared in less than four days at O~C and decreased to one-third over a period of five days on storage at liquid nitrogen temperatures . Neither ethylenediaminetetracetic acid nor mercaptoethanol assisted in preserving However, the enzyme was not as unstable when
the activity of the stored enzyme .
kept as the homogenate, and if the homogenate was kept in the presence of substrate at -80~C, 80-g0% of the activity remained after one week .
The protective
action of substrate was further indicated by the fact that preincubation of the enzyme without substrate for ten minutes destroyed approximately half the activity . Table 2 lists the effects of different compounds on the conversion of tyrosine to catecholamines by the caudate enzyme .
The ratio of Dopa to dopamine
in the Table gives an estimate of the relative effects on Dopa decarboxytase . As can be seen from the Table, under control conditions the activity dopamine is 2g times that
in Dopa,
in
indicating that the decarboxylase activity of
the system was sufficient to convert nearly all the Dopa formed to dopamine . The Dopa decarboxylase activity could be greatly reduced, however,
by 10 -4 M
NSD-1034, with only slight loss
This result
in tyrosine hydroxylase activity .
is similar to that of Nagatsu et al . and of Musacchio and Goldstein
inhibitor
Also, in confirmation of these authors,
(7), p-fluorophenylalanine was found to be a potent
inhibitor of tyrosine hydroxylase . just as potent an
(1) .
However, phenylalanine itself was found to be
in this study, suggesting that the effect of p-fluoro-
phenylalanine may be due to the unsubstituted phenylalanine moiety .
Phenyl-
pyruvate was found to be roughly one-third as potent, while phenyllactic acid was inactive,
It seems possible that phenylpyruvate may assert
amination to phenylalanine, since Fonnum et amination
al . (8)
its effect by trans-
reported reversible trans-
in brain tissue .
The possibility that the inhibiting effect of phenylalanine on tyrosine hydroxylase contributes to the pathology in phenylketonuria needs to be considered .
It
is consistent with the report of Nadier and Hsia (9) that phenylketonurics
have reduced
levels of catecholamines in the plasma and urine which return to
Vol . 3, No. 10
SOME PROPERTIES OF TYROSINE HYDROXYLASE
119 9
TABLE 2 Effect of Various Agents on Conversion of Tyrosine to Catecholamines Final concentration x 10 - 5 M
Compound Control B 1 aalt NSfl-1034 DL-p-Fluorophenylalanine DL-Phenylalanine DL-Phenylalartine + NSD-1034 Phenylpyruvate Phenyllactate L-Dopa L-alpha-Methyldopa L-Tryptophan DL-S-Hydroxytryptophan
DL-atpha-Methylmetatyrosine Dopamide Pyrogallol Chlorpromazine Reserpine Iproniazid Thyroxine Imipramine Lysergic Acid Diethyiamide Heroin Amphetamine Pargyline
10 10 2,5 10 2 .5 10 each 10 2 .5 10 2 .5 0,6 2,5 0 .6 10 2 .5 10 2 .5 10 2,5 2,5 5 10 37 l0 10 10 10 2,8 10 40 l0 10 2 .5
Conversion to GoPamine cpm catecholamines Dopa cpm per cent of control 100 3 80, 90 17, 24 42 24, 19 37 16 61, 50 71 111, 101 53 66 68, 64 73 35 57 45 79 36, 41 68 111, g2 105 126, 93 73 160, 122 13i, 101 115 106, 81 i34, 111 129, 89 108 146, 109 153 145
28 .6 0 .3 4 .7 2,9 0,3 21
0 .7
2,1 1 .1 17
11
normal on a low phenylalanine diet, although plasma tyrosine levels remain unchanged . L-Dopa has a considerable effect
indicating product inhibition of the re-
action .
L-alpha-Methyldopa is comparable in potency, suggesting a similar mode of
action,
The well-known effect of L-alpha-methyldopa on Dopa decarboxylase is also
indicated by the shift
in the dopamine :Dopa ratio from 29 to 0 .3 .
DL-alpha-Methyl-
metatyrosine also inhibited both tyrosine hydroxylase and Dopa decarboxylase,
1200
Vol . 3, No. 10
SOME PROPERTIES OF TYROSINE HYDROXYLASE Tryptophan was almost as active as phenylalanine in
inhibiting the reaction,
but 5-hydroxytryptophan was weaker . A number of the commonly used psychoactive drugs were without influence on the system, but pargyiine, a monoamine oxidase inhibitor, appeared to have some stimulant action, A number of clinically useful drugs are known to influence catecholamine metabolism . step
Since the hydroxylation of tyrosine appears to be the rate-limiting
in catechol~nine synthesis, a study of those compounds
influencing tyrosine
hydroxylase might be particularly fruitful from the point of view of finding useful
new drugs, Acknowledyements The authors are grateful to Dr, E . G . McGeer for valuable advice and to
Dr . D . J . Drain of Smith and Nephew Research for supplying NSD-1034,
This res-
earch was supported by Medical Research Council of Canada Grant #MA-1421, Canad ian Federal-Provincial Mental Health Grant #609 -5 - 141, and a Canadian Mental Health Association grant . References l,
T . Nagatsu, M .
Levitt and S . Udenfriend, Biochem, Biophys . Res . Comet .
14, 543
(1964) . 2.
E .G . McGeer,
G .M .
Ling and P . L . McGeer, Biochem . Biophys . Res . Come . ~, 291
(1963) . 3.
D .T, Masuoka, H .F . 73
Schott and L . Petriello, J . Pharmacol . Exp . Therap . ~,
(1963) .
4.
E .G . McGeer and P . L, McGeer, Can . J . eiochem . 40,
5.
E . G . McGeer and W,H . Clark, J . Chromatog .
6.
C .H . Wang and D .E .
7.
J .M . Musacchio and M . Goldstein, Biochem. Pharmacol .
8.
F . Fonnum, R . Haavaldsen and 0 . Tangen, J . Neurochem .
hl ,
9.
H .L . Nadler and D,Y . Hsia, Proc . Soc . Exp . Biol . Med ,
l~,
14,
1141
(1962),
107 (1964) .
Jones, Biochem, Biophys, Res . Comet ,
l, 203
12,
1061
(1959) . (1963) .
109 (1964) . 721
(1961),
SOME PROPERTIES OF TYROSINE HYOROXYLASE FROM THE CAUDATE NUCLEUS S . P . Bagchi and P .
L . McGeer
Kinsmen Laboratory of Neurological Research, The University of British Columbia, Vancouver, Canada
(Received 4 September 1964)
The least understood step
in the biosynthesis of the catecholamines is
the conversion of tyrosine to 3,4-dihydroxyphenylalanine (Dopa) . investigators have demonstrated, takes place, Nagatsu et al . tions .
Although many
both in vivo and in vitro , that this conversion
(1) were the first to do so using cell-free prepara-
They described some of the properties of tyrosine hydroxylase obtained
from brain stem and other organs . In brain, the caudate nucleus has the highest concentration of dopamine . Moreover,
it has been found to be quite active in converting tyrosine to catech-
olamines both
in vivo (2) and
in vitro (3) .
This report deals with the partial
purification of tyrosine hydroxylase from beef and rat caudate nucleus, some properties of the enzyme, and the effect of several compounds on
its activity .
Methods Beef brain, obtained from a slaughterhouse, was thoroughly chilled within twenty minutes of killing in
ice cold, physiological saline .
rapidly rgnoved from decapitated animals .
Rat brain was
Dissection of the caudates and aii
subsequent steps were performed at 0-S~C . Caudate tissue was homogenized
in ten volumes of
isotonic sucrose .
Whole
homogenate was either incubated at this stage, or subJected to centrifugation . 1195
1196
SOME PROPERTIES OF TYROSINE HYDROXYLASE
Vol . 3, No . 10
Cellular debris and nuclear material were removed by centrifuging at 600 G for ten minutes .
In the case
of
rat tissue, the supernatant was then centrifuged
at 20,000 G for fifteen minutes ;
in the case of beef tissue the supernatant was
centrifuged for a similar time at 12,000 G, pended
in
The sedimented fraction was resus-
its original volume of 0 .1 M phosphate buffer, pH
were treated by precipitation with
60'6
6.4 .
Beef particles
saturated ammonium sulfate .
The protein
precipitate was centrifuged off, and used as the enzyme source . Incubations were carried auf
in air at 37~C for one hour,
Each 25 ml
Erlenmeyer flask contained 270,000 cpm of L-tyrosine-U-C 14 ( .67 millimicromoles, specific activity
360
ing approximately
60
me/mM} ; 2,0 ml of 0 .1 M phosphate buffer, pH
6,4,
contain
mg of protein from the ammonium sulfate precipitation, or
alternatively the equivalent of 280 mg of wet tissue; and 0 .1 ml of an aqueous solution of ical
inhibitor or 0 .1 ml
of
water .
The blank in each case was an ident-
incubation except that the solution containing the enzyme was heated to
$O~C for ten minutes prior to incubation, The reaction was stopped by " adding 2 ml of 0 .8 N perchioric acid . solution was homogenized, centrifuged, and the catecholamines
The
in the supernatant
separated on an alumina column (4) . An aliquot of the column eluant was counted in a liquid scintillation spectrometer . parallel
The counts
in the test eluant minus those in the eluant from the
incubation with inactivated enzyme were taken as a measure of the rela
tive conversion of tyrosine to catechols by tyrosine hydroxylase . In a number of cases, a further aliquot of the eluant was subjected to paper chromatography to separate the products,
Approximately 20 micrograms of
cold carrier Dopa, dopamine, noradrenaline, dihydroxyphenylacetic acid and tyrosine were added and the mixture chromatographed in n-butanol 1N HC1 for
26
hours
(5) .
saturated with
The various areas were cut out and counted directly
in a liquid scintillation spectrometer containing 80-90% of the total
(6) .
Dopa-dopamine formed a single spot
activity on the strip,
The remainder was
tyrosine (6-10%), noradrenai3ne (4-5%} and dihydroxyphenylacetic ac td
in
(0-2°~) .
Vol . 3, No. 10
SOME PROPERTIES OF TYROSINE HYDROXYLASE
119 7
In cases where it was desired to separate Dopa and dopamine, a separate aliquot was chromatographed
in n-butanoi saturated with iN fiCi as before, the
area containing Dopa-dopamine eluted with an ethanol/ .03N HC1 mixture (3 :1), and the eluant respotted and run in methanol/n-butanoi/benzene/water (2 :1 :1 :1) . This system separates Dopa from dopamine (5) .
The areas containing these com-
pounds were cut from the paper and counted as before, Results and Discussion A preliminary experiment with rat caudate indicated that the major part of the enzyme activity could be located in the 20,000 G particulate fraction (Table 1),
A lower speed of centrifugation was employed for separating beef TABLE 1
Formation of Catecholamines from Tyrosine by Various Brain Fractions
Species
Preparation
Catecholamines formed per gm wet tissue per hr mN moles
Rat s`
Beef`
cpm
Whole homogenate
2 .23
10 .4x104
Supernatant
0 .06
0 .3x104
Particulate (20,000 G)
1 .69
7 .9x104
Whole homogenate
0 .97
10 .3x104
Supernatant
0 .33
3 .6x104
Particulate (12,000 G}
0 .63
6 .7x104
~ L-tyrosine used : 0 .25 pc at 3,Oxi0 -6 M final concentration ~ L-tyrosine used : 0,25 pc at 3,ßc10 - 7 M final concentration caudate fractions which might account for the higher activity in the supernatant using this tissue,
The ammonium sulfate precipitate from beef caudate showed a
two-fold increase in activity over the particulate fraction on a protein basis, The enzyme proved to be extr~nely unstable .
The activity of the ammonium
119 8
SOME PROPERTIES OF TYROSINE HYDROXYLASE
Vol . 3, No . 10
sulfate precipitate disappeared in less than four days at O~C and decreased to one-third over a period of five days on storage at liquid nitrogen temperatures . Neither ethylenediaminetetracetic acid nor mercaptoethanol assisted in preserving However, the enzyme was not as unstable when
the activity of the stored enzyme .
kept as the homogenate, and if the homogenate was kept in the presence of substrate at -80~C, 80-g0% of the activity remained after one week .
The protective
action of substrate was further indicated by the fact that preincubation of the enzyme without substrate for ten minutes destroyed approximately half the activity . Table 2 lists the effects of different compounds on the conversion of tyrosine to catecholamines by the caudate enzyme .
The ratio of Dopa to dopamine
in the Table gives an estimate of the relative effects on Dopa decarboxytase . As can be seen from the Table, under control conditions the activity dopamine is 2g times that
in Dopa,
in
indicating that the decarboxylase activity of
the system was sufficient to convert nearly all the Dopa formed to dopamine . The Dopa decarboxylase activity could be greatly reduced, however,
by 10 -4 M
NSD-1034, with only slight loss
This result
in tyrosine hydroxylase activity .
is similar to that of Nagatsu et al . and of Musacchio and Goldstein
inhibitor
Also, in confirmation of these authors,
(7), p-fluorophenylalanine was found to be a potent
inhibitor of tyrosine hydroxylase . just as potent an
(1) .
However, phenylalanine itself was found to be
in this study, suggesting that the effect of p-fluoro-
phenylalanine may be due to the unsubstituted phenylalanine moiety .
Phenyl-
pyruvate was found to be roughly one-third as potent, while phenyllactic acid was inactive,
It seems possible that phenylpyruvate may assert
amination to phenylalanine, since Fonnum et amination
al . (8)
its effect by trans-
reported reversible trans-
in brain tissue .
The possibility that the inhibiting effect of phenylalanine on tyrosine hydroxylase contributes to the pathology in phenylketonuria needs to be considered .
It
is consistent with the report of Nadier and Hsia (9) that phenylketonurics
have reduced
levels of catecholamines in the plasma and urine which return to
Vol . 3, No. 10
SOME PROPERTIES OF TYROSINE HYDROXYLASE
119 9
TABLE 2 Effect of Various Agents on Conversion of Tyrosine to Catecholamines Final concentration x 10 - 5 M
Compound Control B 1 aalt NSfl-1034 DL-p-Fluorophenylalanine DL-Phenylalanine DL-Phenylalartine + NSD-1034 Phenylpyruvate Phenyllactate L-Dopa L-alpha-Methyldopa L-Tryptophan DL-S-Hydroxytryptophan
DL-atpha-Methylmetatyrosine Dopamide Pyrogallol Chlorpromazine Reserpine Iproniazid Thyroxine Imipramine Lysergic Acid Diethyiamide Heroin Amphetamine Pargyline
10 10 2,5 10 2 .5 10 each 10 2 .5 10 2 .5 0,6 2,5 0 .6 10 2 .5 10 2 .5 10 2,5 2,5 5 10 37 l0 10 10 10 2,8 10 40 l0 10 2 .5
Conversion to GoPamine cpm catecholamines Dopa cpm per cent of control 100 3 80, 90 17, 24 42 24, 19 37 16 61, 50 71 111, 101 53 66 68, 64 73 35 57 45 79 36, 41 68 111, g2 105 126, 93 73 160, 122 13i, 101 115 106, 81 i34, 111 129, 89 108 146, 109 153 145
28 .6 0 .3 4 .7 2,9 0,3 21
0 .7
2,1 1 .1 17
11
normal on a low phenylalanine diet, although plasma tyrosine levels remain unchanged . L-Dopa has a considerable effect
indicating product inhibition of the re-
action .
L-alpha-Methyldopa is comparable in potency, suggesting a similar mode of
action,
The well-known effect of L-alpha-methyldopa on Dopa decarboxylase is also
indicated by the shift
in the dopamine :Dopa ratio from 29 to 0 .3 .
DL-alpha-Methyl-
metatyrosine also inhibited both tyrosine hydroxylase and Dopa decarboxylase,
1200
Vol . 3, No. 10
SOME PROPERTIES OF TYROSINE HYDROXYLASE Tryptophan was almost as active as phenylalanine in
inhibiting the reaction,
but 5-hydroxytryptophan was weaker . A number of the commonly used psychoactive drugs were without influence on the system, but pargyiine, a monoamine oxidase inhibitor, appeared to have some stimulant action, A number of clinically useful drugs are known to influence catecholamine metabolism . step
Since the hydroxylation of tyrosine appears to be the rate-limiting
in catechol~nine synthesis, a study of those compounds
influencing tyrosine
hydroxylase might be particularly fruitful from the point of view of finding useful
new drugs, Acknowledyements The authors are grateful to Dr, E . G . McGeer for valuable advice and to
Dr . D . J . Drain of Smith and Nephew Research for supplying NSD-1034,
This res-
earch was supported by Medical Research Council of Canada Grant #MA-1421, Canad ian Federal-Provincial Mental Health Grant #609 -5 - 141, and a Canadian Mental Health Association grant . References l,
T . Nagatsu, M .
Levitt and S . Udenfriend, Biochem, Biophys . Res . Comet .
14, 543
(1964) . 2.
E .G . McGeer,
G .M .
Ling and P . L . McGeer, Biochem . Biophys . Res . Come . ~, 291
(1963) . 3.
D .T, Masuoka, H .F . 73
Schott and L . Petriello, J . Pharmacol . Exp . Therap . ~,
(1963) .
4.
E .G . McGeer and P . L, McGeer, Can . J . eiochem . 40,
5.
E . G . McGeer and W,H . Clark, J . Chromatog .
6.
C .H . Wang and D .E .
7.
J .M . Musacchio and M . Goldstein, Biochem. Pharmacol .
8.
F . Fonnum, R . Haavaldsen and 0 . Tangen, J . Neurochem .
hl ,
9.
H .L . Nadler and D,Y . Hsia, Proc . Soc . Exp . Biol . Med ,
l~,
14,
1141
(1962),
107 (1964) .
Jones, Biochem, Biophys, Res . Comet ,
l, 203
12,
1061
(1959) . (1963) .
109 (1964) . 721
(1961),