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Inhibition of l -tyrosine decarboxylase activity by l -dopa melanin in vitro
ht. 3. Biochem., 1974, Vol. 5, pp. 85 to 87 Pergamon Press. Printed in Great Britain
INHIBITION
OF BY
L-TYROSINE L-DOPA
DECARBOXYLASE PvIELANIN
IjV
85
ACTIVITY
VITRO
NGO T&W Department of Nuclear Medicine
and Radiobiology, Centre Hospitalier Universitaire, Sherbrooke, Quebec, Canada (Received 30 April 1973)
ABSTRACT L-Tyrosine decarboxylase apoenzym e of &reptvcvcauj%ecaiti can easily decarboxylate dihydroxyphenylalanine and this aromatic ~-amino acid decarboxylase is a pyridoxal phosphate-dependent enzyme. 2. The activity of this enzyme involved in the decarboxylation of dihydroxyphenylalanine is continuously inhibited by dihydroxyphenylalanine melanin. I.
IN a recent study, we reported that activities of dopa decarboxylases of the rat liver as well as L-phenylalanine decarboxylase of Streptococcus fadis were inhibited by a product of dihydroxyphenylalanine (dopa),
i.e. L-dopa melanin (Tran & Laplante, 1972). This experiment is a further evaluation of such an inhibition by L-dopa melanin on bacterial decarboxylase apoenzyme involved in the decarboxylation of dopa in vitro.
, 7 6
051mg L-DOPAmelanin 0
0
15
30
45
,OmM PLP
60 75 Time, min.
90
105
120
FIG. I.-Composite data of rates of WO, production from dopa-carboxyl-W incubated with or without L-tyrosine decarboxylase apoenzyme, PLP, and ~-dopa melanin. The ordinate represents per cent of incubated l*C produced as WZOs per minute and the abscissa represents the time in minutes after administration of dopa-carboxyl-1%. Each point represents the mean of WO, production for each group of experiments at the given time; the length of the vertical bar through each point represents + one standard error of the mean.
86
NC0 TRAN
MkTERIALS AND METHODS Details of the apparatus devised for an instantaneous and continuous measurement of dopa decarboxylase activity have been published recently (Tran, 1972). For each study, 4 mg. L-tyrosine decarboxylase apocnzyme of Strtptmmu foccalis (Sigma Chemical Co., St. Louis, MO., U.S.A.) were incubated with 024 t&i 3,4&hydroxyphenyIalanine-~~l-l*C(dopa-~~~l-l4C) (specific activity: 3’4 mCi~mmoie; New England Nuclear Co., Boston, Mass., U.S.A.) with or without pyridoxal phosphate (PLP) (Nutritional Biochemical Co., Cleveland, Ohio, U.S.A.) and ~-dopa melanin in o’ IM phosphate buffer, pH 70, at 37” C, in an atmosphere of nitmgen. L-dopa melanin was prepared by an aut0xidation of Ldopa incubated in o-rM phosphate buffer, pH 70, at 37” C, and gassed with oxygen for 12 hours (Mason, Ingram & Allen, 1960; Horak & Gilette, rg7r ; Tran & Laplante, x972).
FIG. I represents curves depicting rates of %O, production from dopa-carboxyl-1% when this substrate was incubated with L-tyrosine dccarboxylase apocuzyme and PLP, with or without Ldopa melanin. For comparing various curves, we determined cumulative WO; production within I 20 minutes. This parameter has been defined previously (Tran & Laplante, 1972; Tran, rg72a). As shown in Tables I and II, an increased r*COs production (PC 0.05) was obtained when larger quantities of PLP was added to the same amounts of L-tyrosine decarboxylase apoenzyme and 14C-Iabeled dopa. However, a decreased WO, production (P c o’oor ) was noted after administration of 0.515 mg. Ldopa melanin to the incubation chamber. A complete inhibition of dopa decarboxylation was seen with 5. s5 mg. i-dopa melanin. Table I.-CWLA~~E
WOI PRODU~I-ION FROM INCUBATEDWITH L-TYROSIRE
DRCARR~X~O~R APOENZVMEAND TWO CONCENTRATIoNsOF PLP (kl-R00RY (The number of determinations is in parentheses)
4 mg. apoenxymc+ o*rmM PLP (4) 4 mg. apoenxyme+ IYXXIMPLP (4)
Table IL-C
uhfux.A-rrvRldCO, PR0no0r10~ ~Robi
DOPA-CARBOXYL-1% IXUBA-I-ED wmi L-TYROSINE ~CARBOXyLAsE APOENZYME, PLP, AND VARIOUS ~ONClWIXATIONS OF L-DOPAMELANIN
14CPRODUCTION wrrnx~
CATJXWRY
(The number of determinations is in parentheses) 4 mg. apoenayme+ ~*ornM PLP (4) 4 ~g..~Lff”f
120 MxNuTRs
(Per cent+ S-E.) 2’577 ?1:0’434 1*648~0~074(P
+0515 mg. L-dopa melanin (4) 4 mg. ap0enzyme + r*omM PLP+5*150 mg. x.-dopa melanin (4)
0
DISCUSSION
RESULTS
DOPA-~RBOXYL~C
Int. J. Biochm.
W PR0m~cr10~ wrrmR 12omin.
(Per cent + S.E.)
2’3772 0’302 3.895+0*310(Pco.05)
The conversion of dopa to dopamine is made by aromatic amino acid decarboxylases of animal(Sourkes, I 966; Sandier & Ruthven, 1969) and human (Lloyd & I-Iomykiewicz, 1970; Vogel, MacFarland & Prince, 1970) tissues. We recently found that such a conversion was also made by several bacterial and Lenzymes, e.g., t-phenyh&nine tyrosine decarboxylases of Stre~dococcas faecalr; (Tran, 1971) and L-histidine decarboxylase of Clostridirm wclchii (Tran, xg72b). The results presented here show that L-tyrosine decarboxyiasc apoenzyme of S~e~~ococcus faecdis can easily decarboxylate dopa in an atmosphere of nitrogen. This amino acid decarboxylase is a PLP-dependent enzyme, due to the fact that changes in cumulative ‘*CO, production within 120 minutes were seen following administration of larger amounts of PLP to the incubation chamber. Similar data were found with bacterial L-phenylalanine (Tran, I 97 I 1, L-tyrosine (Tran, rg7r), and L-histidine (Tran, rg72b) decarboxylases. These overall results suggest that the production of dopamine in the brain may not be due to a specific dopa decarboxylase only, but also to many other amino acid decarboxylases. Interestingly, we found that the activity of L-tyrosine decarboxylase apoenzyme involved in the decarboxylation of dopa was inhibited by a product obtained from the autoxidation of dopa, i.e. L-dopa melanin (Tran & Laphtnte, 1972).
‘9743 5
L-TYROSINE
DECARBOXYLASE
REFERENCES HORAK, V., 8; GILETTE,J. R. (rg71), ‘ A study of the oxidation-reduction state of synthetic ~,+dihydro-nL-phenylalanine melanin ‘, Mol. %kM;O~, 7, 42-j-&. LLOYD. K.. & HORNYIUEWXZ. 0. (ISlO). ‘ Paknsoh’s disease: activity of L-dopa decarboxylase in discrete brain regions ‘, Sctencc,.N.Y., 170, 1212-1213. MAX)N, H. S., INGRAM, D. J. E., & ALLEN, B. ( f 960)) ‘ The free radical property of melanins ‘, Archs. Biochem. Biophys., 86, 225-230. SANDLER, M., & Ru-mvz N, c. R.J. (K&J), ‘ The biosynthesis and metabolism of the catecbolamines ‘, in Progress in Medicinal Chcrnistsy (cd. Ellis & West), pp 200-265. London: Butterworths. SOURKES,T. L. (Ig66), ‘ L-dopa decarboxylases: substrates, coenzymes, inhibitors ‘, Phatmacol. R&v., 18,53-b ‘ Interaction of aromatic TM, NGo (Ig71), L-amino-acid decarboxylases with pyridoxal phosphate ‘, Znt.3. Biochcm., zz, 700-704.
ACTMTY
87
TM, NGO (Ig72a), ‘ Improved ionization chamber method for continuous measurement of dopa decarboxylase activity ‘, Ann&. Biochcm., 48, I 12-1 Ig. TM, NGO (Ig72b), ‘ Patterns of enzymatic and non-enzymatic oxidation of dopa in vitro ‘, 3. J%&. Med., X3, 34CJ-352. TRAN, NGO, & LAP-, M. (19721, ‘ Inhibition of aromatic L-amino acid decarboxylase activities by ~.4-dihvdroxvphenylalanine melanin ‘, Int. j.OB&-bm.,‘g, II~-122: VOGEL. W. H.. MACFARLAND. H.. & PRINCE. L. X’. ( IgTo), ” Decarboxylatik ,oi 3,4-dihydro: xyphenylalanine in various human adult and fetal tissues‘, Biochem. Pharnuu., ~g, 618-620.
Key Word Zndex: L-Tyrosine decarboxylase, L-dopa, melanin, Streptococczfs facalis.
INHIBITION
OF BY
L-TYROSINE L-DOPA
DECARBOXYLASE PvIELANIN
IjV
85
ACTIVITY
VITRO
NGO T&W Department of Nuclear Medicine
and Radiobiology, Centre Hospitalier Universitaire, Sherbrooke, Quebec, Canada (Received 30 April 1973)
ABSTRACT L-Tyrosine decarboxylase apoenzym e of &reptvcvcauj%ecaiti can easily decarboxylate dihydroxyphenylalanine and this aromatic ~-amino acid decarboxylase is a pyridoxal phosphate-dependent enzyme. 2. The activity of this enzyme involved in the decarboxylation of dihydroxyphenylalanine is continuously inhibited by dihydroxyphenylalanine melanin. I.
IN a recent study, we reported that activities of dopa decarboxylases of the rat liver as well as L-phenylalanine decarboxylase of Streptococcus fadis were inhibited by a product of dihydroxyphenylalanine (dopa),
i.e. L-dopa melanin (Tran & Laplante, 1972). This experiment is a further evaluation of such an inhibition by L-dopa melanin on bacterial decarboxylase apoenzyme involved in the decarboxylation of dopa in vitro.
, 7 6
051mg L-DOPAmelanin 0
0
15
30
45
,OmM PLP
60 75 Time, min.
90
105
120
FIG. I.-Composite data of rates of WO, production from dopa-carboxyl-W incubated with or without L-tyrosine decarboxylase apoenzyme, PLP, and ~-dopa melanin. The ordinate represents per cent of incubated l*C produced as WZOs per minute and the abscissa represents the time in minutes after administration of dopa-carboxyl-1%. Each point represents the mean of WO, production for each group of experiments at the given time; the length of the vertical bar through each point represents + one standard error of the mean.
86
NC0 TRAN
MkTERIALS AND METHODS Details of the apparatus devised for an instantaneous and continuous measurement of dopa decarboxylase activity have been published recently (Tran, 1972). For each study, 4 mg. L-tyrosine decarboxylase apocnzyme of Strtptmmu foccalis (Sigma Chemical Co., St. Louis, MO., U.S.A.) were incubated with 024 t&i 3,4&hydroxyphenyIalanine-~~l-l*C(dopa-~~~l-l4C) (specific activity: 3’4 mCi~mmoie; New England Nuclear Co., Boston, Mass., U.S.A.) with or without pyridoxal phosphate (PLP) (Nutritional Biochemical Co., Cleveland, Ohio, U.S.A.) and ~-dopa melanin in o’ IM phosphate buffer, pH 70, at 37” C, in an atmosphere of nitmgen. L-dopa melanin was prepared by an aut0xidation of Ldopa incubated in o-rM phosphate buffer, pH 70, at 37” C, and gassed with oxygen for 12 hours (Mason, Ingram & Allen, 1960; Horak & Gilette, rg7r ; Tran & Laplante, x972).
FIG. I represents curves depicting rates of %O, production from dopa-carboxyl-1% when this substrate was incubated with L-tyrosine dccarboxylase apocuzyme and PLP, with or without Ldopa melanin. For comparing various curves, we determined cumulative WO; production within I 20 minutes. This parameter has been defined previously (Tran & Laplante, 1972; Tran, rg72a). As shown in Tables I and II, an increased r*COs production (PC 0.05) was obtained when larger quantities of PLP was added to the same amounts of L-tyrosine decarboxylase apoenzyme and 14C-Iabeled dopa. However, a decreased WO, production (P c o’oor ) was noted after administration of 0.515 mg. Ldopa melanin to the incubation chamber. A complete inhibition of dopa decarboxylation was seen with 5. s5 mg. i-dopa melanin. Table I.-CWLA~~E
WOI PRODU~I-ION FROM INCUBATEDWITH L-TYROSIRE
DRCARR~X~O~R APOENZVMEAND TWO CONCENTRATIoNsOF PLP (kl-R00RY (The number of determinations is in parentheses)
4 mg. apoenxymc+ o*rmM PLP (4) 4 mg. apoenxyme+ IYXXIMPLP (4)
Table IL-C
uhfux.A-rrvRldCO, PR0no0r10~ ~Robi
DOPA-CARBOXYL-1% IXUBA-I-ED wmi L-TYROSINE ~CARBOXyLAsE APOENZYME, PLP, AND VARIOUS ~ONClWIXATIONS OF L-DOPAMELANIN
14CPRODUCTION wrrnx~
CATJXWRY
(The number of determinations is in parentheses) 4 mg. apoenayme+ ~*ornM PLP (4) 4 ~g..~Lff”f
120 MxNuTRs
(Per cent+ S-E.) 2’577 ?1:0’434 1*648~0~074(P
+0515 mg. L-dopa melanin (4) 4 mg. ap0enzyme + r*omM PLP+5*150 mg. x.-dopa melanin (4)
0
DISCUSSION
RESULTS
DOPA-~RBOXYL~C
Int. J. Biochm.
W PR0m~cr10~ wrrmR 12omin.
(Per cent + S.E.)
2’3772 0’302 3.895+0*310(Pco.05)
The conversion of dopa to dopamine is made by aromatic amino acid decarboxylases of animal(Sourkes, I 966; Sandier & Ruthven, 1969) and human (Lloyd & I-Iomykiewicz, 1970; Vogel, MacFarland & Prince, 1970) tissues. We recently found that such a conversion was also made by several bacterial and Lenzymes, e.g., t-phenyh&nine tyrosine decarboxylases of Stre~dococcas faecalr; (Tran, 1971) and L-histidine decarboxylase of Clostridirm wclchii (Tran, xg72b). The results presented here show that L-tyrosine decarboxyiasc apoenzyme of S~e~~ococcus faecdis can easily decarboxylate dopa in an atmosphere of nitrogen. This amino acid decarboxylase is a PLP-dependent enzyme, due to the fact that changes in cumulative ‘*CO, production within 120 minutes were seen following administration of larger amounts of PLP to the incubation chamber. Similar data were found with bacterial L-phenylalanine (Tran, I 97 I 1, L-tyrosine (Tran, rg7r), and L-histidine (Tran, rg72b) decarboxylases. These overall results suggest that the production of dopamine in the brain may not be due to a specific dopa decarboxylase only, but also to many other amino acid decarboxylases. Interestingly, we found that the activity of L-tyrosine decarboxylase apoenzyme involved in the decarboxylation of dopa was inhibited by a product obtained from the autoxidation of dopa, i.e. L-dopa melanin (Tran & Laphtnte, 1972).
‘9743 5
L-TYROSINE
DECARBOXYLASE
REFERENCES HORAK, V., 8; GILETTE,J. R. (rg71), ‘ A study of the oxidation-reduction state of synthetic ~,+dihydro-nL-phenylalanine melanin ‘, Mol. %kM;O~, 7, 42-j-&. LLOYD. K.. & HORNYIUEWXZ. 0. (ISlO). ‘ Paknsoh’s disease: activity of L-dopa decarboxylase in discrete brain regions ‘, Sctencc,.N.Y., 170, 1212-1213. MAX)N, H. S., INGRAM, D. J. E., & ALLEN, B. ( f 960)) ‘ The free radical property of melanins ‘, Archs. Biochem. Biophys., 86, 225-230. SANDLER, M., & Ru-mvz N, c. R.J. (K&J), ‘ The biosynthesis and metabolism of the catecbolamines ‘, in Progress in Medicinal Chcrnistsy (cd. Ellis & West), pp 200-265. London: Butterworths. SOURKES,T. L. (Ig66), ‘ L-dopa decarboxylases: substrates, coenzymes, inhibitors ‘, Phatmacol. R&v., 18,53-b ‘ Interaction of aromatic TM, NGo (Ig71), L-amino-acid decarboxylases with pyridoxal phosphate ‘, Znt.3. Biochcm., zz, 700-704.
ACTMTY
87
TM, NGO (Ig72a), ‘ Improved ionization chamber method for continuous measurement of dopa decarboxylase activity ‘, Ann&. Biochcm., 48, I 12-1 Ig. TM, NGO (Ig72b), ‘ Patterns of enzymatic and non-enzymatic oxidation of dopa in vitro ‘, 3. J%&. Med., X3, 34CJ-352. TRAN, NGO, & LAP-, M. (19721, ‘ Inhibition of aromatic L-amino acid decarboxylase activities by ~.4-dihvdroxvphenylalanine melanin ‘, Int. j.OB&-bm.,‘g, II~-122: VOGEL. W. H.. MACFARLAND. H.. & PRINCE. L. X’. ( IgTo), ” Decarboxylatik ,oi 3,4-dihydro: xyphenylalanine in various human adult and fetal tissues‘, Biochem. Pharnuu., ~g, 618-620.
Key Word Zndex: L-Tyrosine decarboxylase, L-dopa, melanin, Streptococczfs facalis.