ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 191, No. 2, December, pp. 714-718, 1978
Multiple Catalytic Sites of Rat Brain Mitochondrial TORU
EGASHIRA,*
YUKIO
KUROIWA,*
AND
Monoamine KAZUYA
Oxidase
KAMIJO$
‘Department of Biochemical Toxicology School of Pharmaceutical Sciences and* Department of Pharmacology, School of Medicine, Showa University, Tokyo 142 Received February
22, 1978
We compared the inhibitory and cat&tic effects of various monoamines on forms A and B of monoamine oxidase (MAO) on mitochondrial preparations from rat brain in mixed substrate experiments. MAO activity was determined by a radioisotopic assay. MAO showed than for tyramine and serotonin. lower K,,, values for tryptamine and /?-phenylethylamme The K,,, values of the untreated preparation for tyramine, tryptamine, and P-phenylethylamine obtained were the same as those of the form B enzyme and the K, value for serotonin was the same as that of the form A enzyme. Tyramine and tryptamine were competitive inhibitors of serotonin oxidation and /l-phenylethylamine did not bind with form A enzyme or inhibit the oxidation of serotonin, while tyramine and tryptamine were competitive inhibitors of /I-phenylethylamine oxidation. Although serotonin was not oxidized by form B enzyme, serotonin was a competitive inhibitor of /3-phenylethylamine oxidation. It is suggested that rat brain mitochondrial MAO is characterized by two kinds of binding sites.
Monoamine oxidase (monoamine:oxidoreductase (deaminating) EC 1.4.3.4), which catalyzes the oxidation of biogenic amines such as serotonin and catecholamines, has been found in various mammalian tissues. There is a evidence for the existence of multiple forms of monoamine oxidase, which can be distinguished by their differences in sensitivity to heat treatment (1,2) and inhibitors (3, 4) and in their antigenic properties (5,6) and electrophoretic mobilities (7-9). The physiological significance, however, of the multiple forms of monoamine oxidase is unknown. Johnston (10) demonstrated the existence of two different functional forms of monoamine oxidase (forms A and B) in rat brain, in studies with the drug inhibitor clorgyline (N-methyl-N-propargyl-3-(2,4dichlorophenoxy) -propylamine) : form A is very sensitive to clorgyline but form B is only inhibited by a high concentration of clorgyline. In contrast to clorgyline, the drug deprenyl (phenylisopropyhnethylpropinylamine) (11) inhibits form B at lower concentration than form A. Serotonin and norepinephrine are preferential substrates for form A monoamine oxidase and benxylamine and /3-phenylethylamine are prefer-
ential substrates for form B monoamine oxidase (12). Tyramine is oxidized by both forms (12). In the present study, using mixtures of two substrates, we compared the inhibitory and catalytic effects of various monoamine on forms A and B monoamine oxidase in mitochondrial preparations from rat brain. MATERIALS
Copyright 0 1978 by Academic Press, Inc. AU rights of reproduction in any form reserved.
METHODS
Male Wistar strain rate weighing 150 to 200 g were decapitated and their brains were removed and homogenized in 10 volumes of 0.32 M sucrose. The mitochondrial fraction was prepared by differential centrifugation using the method of Koenig et al. (13). The mitochondria were washed once by resuspending them in 0.32 M sucrose solution and used as the enzyme preparation. Monoamine oxidase activity was estimated essentially as described by Fuller (14) with [“C]tyramine and [‘%]serotonin and by Ekstedt (15) with [“Cltryptamine and P-[“C]phenylethylamine as substrates. The incubation medium contained various concentrations of radioactive substrates in a total volume of 275 d of potassium phosphate buffer, pH 7.2. The reaction was started by adding 25 al of the enzyme preparation. Incubation was carried out for 20 min at 37’C and then the reaction was stopped by adding 0.2 ml of 2 N HCl. With tyramine and serotonin, the reaction products were extracted with ether for 15 s in a homomixer and with tryptamine and /3-phenylethylamine 714
0003-9861/78/1912-0714$02.00/O
AND
MULTIPLE
CATALYTIC
SITES OF MONOAMINE
the products were extracted with toluene for 15 mm in a shaker. Samples of the extracts were mixed with 6 ml of Aquasol as scintillation liquid and their radioactivities were measured with a Packard Tri-Carb Liquid Scintillation Spectrometer. The enzyme activity was expressed in disintegrations per minute (dpm) extracted after 20 min of incubation. The dpm was a linear proportionality during the incubation time. Protein content was estimated by the method of Lowry et al. (16) and protein concentrations of the enzyme preparation were adjusted to 40 mg/ml. Mitochondriel treatments with clorgyline and deprenyl: Mitochondrial preparations were preincubated with either clorgyline (1 w) or deprenyl (10 pM) in a total volume of 1.0 ml at 37°C for 4 h (17), and then the monoamine oxidase activity of samples of the mixtures was estimated. Preparations treated with clorgyline had 98% of the control activity toward pphenylethylamine and 2% of that toward serotonin. Conversely, preparations treated with deprenyl had 2% and 60% of the control activities toward /3-phenylethylamine and serotonin, respectively. Thus, forms A and B were irreversibly inhibited by clorgyline and deprenyl(171, respectively, and in subsequent experiments, preparations treated with deprenyl and clorgyline were used as preparations of form A and B of monoamine oxidase, respectively. Deprenyl was kindly provided by Prof. Knoll, Budapest, Hungary and clorgyline from May & Baker Ltd., Degenham, England.
715
OXIDASE
showed lower K,,, values for tryptamine and P-phenylethylamine than for tyramine and serotonin. The K,,, values of the untreated preparation for tyramine, tryptamine, and /3-phenylethylamine obtained were the same as those of the form B enzyme, and the K,,, value for serotonin was the same as that of the form A enzyme.
Reaction Rates with a Mixture Substrates
of
Two
Figures 1 through 4 show the inhibitions by other substrates of the oxidations of labeled serotonin and P-phenylethylamine with untreated (Figs. 1 and 2), deprenyltreated (Fig. 3), and clorgyline-treated (Fig. 4) preparations. The reaction rate was es-
RESULTS
Determination of K,,, Values The K,,, values of various substrates for the untreated preparation (forms A and B) and the deprenyl-treated (form A) and clorgyline-treated (form B) preparations were calculated from Lineweaver-Burk plots. The results with serotonin, tyramine, tryptamine, and P-phenylethylamine as substrates are shown in Table I. The monoamine oxidase in rat brain
FIG. 1. Inhibition by various monoamines of serotonin oxidation by rat brain mitochondrial monoamine oxidase. Lineweaver-Burk plots of the reciprocal of the initial velocity of serotonin oxidation against the reciprocal of the labeled serotonin concentration in the presence of unlabeled monoamines at concentrations of twice their K, values. X-X, 30 pM tryptamine; A-A, 200 w tyramine; M, 4.6 JLM pphenylethylamine; M, no additional monoamine. The results are means of triplicate assays.
TABLE I K, VALUES FOR MONOAMINE OXIDASE IN UNTREATED, DEPRENYL-TREATED RAT BRAIN MITOCHONDRIA”
Serotonin Tyramine Tryptamine P-Phenylethylamine
Untreated mitochondria (PM) 89 100 15 2.3
Form A* deprenyl-treated mitochondria (PM) 80 51 5
AND CLORGYLINE-TREATED
Form B** clorgylme-treated mitochondria (PM) 120 15 1.6
a Bat brain mitochondria were preincubated with either deprenyl (10 F)* or clorgyline (1 PM)‘* in a total volume of 1.0 ml at 37°C for 4 h (see Materials and Methods) and then monoamine oxidase activity was estimated. K,,, values were obtained from Lineweaver-Burk plots with various concentrations of labeled substrates. The results are means of triplicate assays.
716
EGASHIRA,
KUROIWA,
AND
KAMIJO
I,,-PHE”ILETHILW,YE ‘ , innI-‘, FIG. 2. Inhibition
by various monoamines of pphenylethylamine oxidation of rat brain mitochondrial monoamine oxidase. Lineweaver-Burk plots .of the reciprocal of the initial velocity of /I-phenylethylamine oxidation against the reciprocal of the labeled P-phenylethylamine concentration in the presence of unlabeled monoamines at concentrations of twice their K,,, values. X-X, 30 PM tryptamine; A-A, 200 pM tyramine; M, 178 pM serotonin, u, no additional monoamine. The results are means of triplicate assays.
FIG. 4. Inhibition by various monoamines of pphenylethylamine oxidation by clorgylme-treated mitochondrial monoamine oxidase. Lineweaver-Burk plots of the reciprocal of the initial velocity of /3phenylethylamine oxidation against the reciprocal of the labeled P-phenylethylamine concentration in the presence of unlabeled monoamines at concentrations of twice their Km values. X-X, 30 PM tryptamine; A-A, 240 pM tyramine; o---O, 178 PM serotonm, u, no additional monoamine. The results are means of triplicate assays.
lines crossed at a single point on the ordinate, showing that tyramine and tryptamine are competitive inhibitors of serotonin oxidation. However, the plot of $ vs. 1
-10
IO I, SEROTOWI”
,
( .W’,
FIG. 3. Inhibition by various monoamines of serotonin oxidation by deprenyl-treated mitochondrial monoamine oxidase. Lineweaver-Burk plot of the reciprocal of the initial velocity of serotonin oxidation against the reciprocal of the labeled serotonin concentration in the presence of unlabeled monoamines at concentrations of twice their K,,, values. X-X, 10 PM tryptamine; A-A, 102 pi tryamine; M, 4.6 PM /3-phenylethylamine; M, no additional monoamine. The results are means of triplicate assays.
timated by measuring the radioactivity of the oxidation product. As shown in Figs. 1 and 3, with the untreated or form A preparation, values of i 1 gave straight lines in the [ serotonin] presence and absence of tyramine, tryptamine, and /3-phenylethylamine. These us
in the presence of P-phenyleth[serotonin] ylamine coincided with the control line, indicating that P-phenylethylamine does not bind with form A enzyme or inhibit the exidation of serotonin. These results are understandable since forms A and B do not react with /3-phenylethylamine and serotonin, respectively. Figures 2 and 4 show plots of the reciprocals of the rates of the reaction and of pphenylethylamine concentration for untreated and form B preparations in the presence and absence of tyramine, tryptamine, and serotonin. The data indicate that tyramine and tryptamine are competitive inhibitors of P-phenylethylamine oxidation. Although serotonin was not oxidized by the form B enzyme, the data show that serotonin is a competitive inhibitor of P-phenylethylamine oxidation. Therefore, serotonin seems to bind to the active site of the form B as an inhibitor. DISCUSSION
The Km values of rat brain mitochondrial monoamine oxidase were calculated from
MULTIPLE
CATALYTIC
SITES
OF MONOAMINE
717
OXIDASE
amine oxidase reaction:
Lineweaver-Burk plots with four labeled substrates. P-Phenylethylamine had the lowest Km value for untreated mitochondria and form B monoamine oxidase. The Km value of the untreated preparations for serotonin was the same as that of the form A enzyme, whereas the Km values for tyramine, tryptamine, and /I-phenylethylamine were similar to those of the form B enzyme. The differences in the K,,, values of the forms A and B for tyramine and tryptamine were comparable. These findings suggest that these substrates are oxidized by both forms of mitochondrial monoamine oxidase, and that when untreated mitochondria are used as an enzyme source, tyramine, tryptamine, and j%phenylethylamine are oxidized predominantly by the form B, although the form A has a higher affinity for these substrates. We measured the oxidation rates of various substrates in the presence of another substrate using untreated mitochondria, and the forms A and B of enzyme from rat brain, because if two substrates are oxidized at the same site, the presence of the one will competitively inhibit oxidation of the other, whereas if they are oxidized by different enzymes, the presence of one should not affect oxidation of the other. When there are two substrates, S1 and Sp, in the reaction mixture, the following mechanism may be proposed for the mono-
KAl EA+ SI +EA&
K1 -EA
+ PI
PI
+ Pz
PI
+ P,
[31
K2’ EeSz Ee + Pz
[41
EA+SZ- KAZ\E&-E+,
K,’
I%+.‘%-xEeSl-EB EB+&-
Ke,,
K2
where EA and Es are the form A and B enzymes, KA,, KAY, KB~, and KB, are the Michaelis constants of the respective reactions and PI and Pz the products. When S1 is serotonin and SP is tyramine or tryptamine, the reaction 3 will not occur, because the form B does not oxidize serotonin. The rate v1 of oxidation of S1 can be expressed as follows:
--=Wll
v1 -
WI1
PI
dt
[Sl] + KAY
When S1 is /3-phenylethylamine and SZ is tyramine or tryptamine, the following equation can be obtained from equations 3 and 4, V’[Sl j Vl = (6) [Sl] + Kg,
TABLE
II
VALUESOF KAp AND KBZ” Enzyme
S*
SP
KAY or I&
&G Untreated Mitochondria (Forms A and B)
Deprenyl-treated chondria (Form A) Clorgyline-treated chondria (Form B)
SERO
89
PEA
2.3
mito-
SERO
80
mito-
PEA
1.6
TYRA TRYPT PEA TYRA TRYPT SERO TYRA TRYPT PEA TYRA TRYPT SERO
100 15 2.3 106 15 89 51 5 2.3 120 15 89
104 7 155 14 320 129 5 113 10 480
’ Rat brain untreated, deprenyl-treated, and clorgyline-treated mitochondria were used as enzyme sources. The concentration of labeled substrates, S, was varied in the presence or absence of another substrate, Sp, at a fiied concentration of twice the K,,, value. K,,, values are quoted from Table I. The apparent Michaelis constants in the presence of Sz were determined from the slopes of double reciprocal plots using the data in Figs. 1-4. SERO, serotonin; TYRA; tyramine; TRYPT, tryptamine; PEA, /I-phenylethylamine. The values are means of triplicate assays.
718
EGASHIRA,KUROIWA,
AND
The KA, and Kg, values were calculated from annarent Michaelis constants, KA, (1 +‘e)
and KB~( 1 +e)
using
KAMIJO REFERENCES
1. YANG, H. Y. T., GORIDIS, C., AND NEFF, N. H. (1972) J. Neurochem. 19, 1241-1250. 2. YANG, H. Y. T., AND NEFF, N. H. (1973) J. Pharmacol. Exp. Ther. 187,365-371. 3. FULLER, R. W. (1972) Adu. Biochem. Psycho-
the data sho& in Figs. l-4. These values obtained (Table II) were similar to those in Table I calculated from Lineweaver-Burk pharmacol. 5,339-354. plots of values in the presence of only one YABUHARA, H. (1974) Japan. J. Pharmacol. 24, substrate. Form B did not oxidize serotonin. 523-533. /&Phenylethylamine did not inhibit the oxMCCAULEY, R., AND RACKER, E. (1973) Mol. Cell. Biochem. 1, 73-81. idation of serotonin, but serotonin inhibited YOUDIM, M. B. H. (1974) Adu. Biochem. Psychothe oxidation of P-phenylethylamine by the pharmacol. l&59-63. untreated preparation or the form B of 7. COLLINS, G. G. S., SANDLER, M., WILLIAMS, E. D., enzyme. AND YOUDIM, M. B. H. (1970) Nature 225, The K,,, value for serotinin was 80-89 817-820. PM with a single substrate, while the Kg, 8. SHIH, J., AND EIDUSON, S. (1971) J. Neurochem. value of this substrate was 320-480 pM (see 18, 1221-1227. Table II). These results show that serotonin 9. JAIN, M. L., AND SANDS, F. L. (1974) J. Neurocan bind to form B enzyme with very weak them. 23, 1291-1293. affinity and not be oxidized by it. 10. JOHNSTON, J. P. (1966) B&hem. Pharmacol. 17, 1285-1297. Tryptamine has been identified in human 11. KNOLL, J., AND MAGYAR, K. (1972) Adu. Biochem. urine (18) and certain tissues, including Psychopharmacol. 5,393-407. brain (19, 20), and it has been reported to be related to etiological factors of schizo- 12. NEFF, N. H., AND YANG, H. Y. T. (1974) Life Sci. 14,2061-2074. phrenia (21), depression (22) and parkin- 13. KOENIG, H., GAINES, D., MCDONALD, T., GRAY, sonism-like symptoms (23). Moreover, R., AND SCOTT, J. (1964) J. Neurochem. 11, tryptamine was recently found in the cen729-740. tral nervous system, suggesting that it may 14. FULLER, R. W. (1968) Arch. Int. Pharmacodyn. act at the same receptor sites as serotonin 174.32-36. in the brain (24). It has also been found in 15. EKSTEDT, B. (1976) Biochem. Pharmacol. 25, 1133-1138. both serotonergic and catecholaminergic 16. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., neurons, suggesting its transmitter function (25).
The comparison between tryptamine and serotonin would presume the strong inhibitory action of tryptamine on serotonin oxidation by untreated mitochondria and form A enzyme from rat brain, and thus it may regulate the metabolism of serotonin. However, tryptamine also strongly inhibits P-phenylethylamine oxidation. Thus, besides binding to forms A and B, tryptamine is a substrate for both forms in the brain. Therefore, studies on the concentration of tryptamine may serve to elucidate the functional significance of monoamine oxidase in the brain. ACKNOWLEDGMENT We are sincerely grateful to Profeeeor Yasuyuki Ogura (Department of Biochemistry, Tchci University) for his helpful discussion and advice.
17. 18. 19.
20. 21.
AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. EGASHIRA, T., EKSTEDT, B., AND ORELAND, L. (1976) B&hem. Pharmacol. 25,2583-2586. PERRY, T. L., AND SCHROEDER, W. A. (1963) J. Chromatogr. 12,368-372. MARTIN, W. R., SLOAN, J. W., CHRISTIAN, S. T., AND CLEMENTS, T. H. (1972) Psychopharmacologia 24,331-346. SAAVEDRA, J. H., AND AXELROD, J. (1972) J. Pharmacol. Exp. Ther. 182,363-369. BRUNE, G. G., AND HIMWICH, H. E. (1962) Arch.
Gen. Psychiatr. 6, 324-346. 22. DEWHURST, W. G. (1968) Nature 218,1130-1133. 23. SAAVEDRA, J. M., HELLER, B., AND FISCHER, E. (1970) Nature 220,868. 24. TEDESCHI, D. H., TEDESCHI, R. E., AND FELLOWS, E. J. (1959) J. PhurntacoZ. Exp. Ther. 126, 223-231. 25. MARSDEN, C. A., AND CURZON, G. (1974) J. Neurochem. 23, 1171-1176.
Multiple Catalytic Sites of Rat Brain Mitochondrial TORU
EGASHIRA,*
YUKIO
KUROIWA,*
AND
Monoamine KAZUYA
Oxidase
KAMIJO$
‘Department of Biochemical Toxicology School of Pharmaceutical Sciences and* Department of Pharmacology, School of Medicine, Showa University, Tokyo 142 Received February
22, 1978
We compared the inhibitory and cat&tic effects of various monoamines on forms A and B of monoamine oxidase (MAO) on mitochondrial preparations from rat brain in mixed substrate experiments. MAO activity was determined by a radioisotopic assay. MAO showed than for tyramine and serotonin. lower K,,, values for tryptamine and /?-phenylethylamme The K,,, values of the untreated preparation for tyramine, tryptamine, and P-phenylethylamine obtained were the same as those of the form B enzyme and the K, value for serotonin was the same as that of the form A enzyme. Tyramine and tryptamine were competitive inhibitors of serotonin oxidation and /l-phenylethylamine did not bind with form A enzyme or inhibit the oxidation of serotonin, while tyramine and tryptamine were competitive inhibitors of /I-phenylethylamine oxidation. Although serotonin was not oxidized by form B enzyme, serotonin was a competitive inhibitor of /3-phenylethylamine oxidation. It is suggested that rat brain mitochondrial MAO is characterized by two kinds of binding sites.
Monoamine oxidase (monoamine:oxidoreductase (deaminating) EC 1.4.3.4), which catalyzes the oxidation of biogenic amines such as serotonin and catecholamines, has been found in various mammalian tissues. There is a evidence for the existence of multiple forms of monoamine oxidase, which can be distinguished by their differences in sensitivity to heat treatment (1,2) and inhibitors (3, 4) and in their antigenic properties (5,6) and electrophoretic mobilities (7-9). The physiological significance, however, of the multiple forms of monoamine oxidase is unknown. Johnston (10) demonstrated the existence of two different functional forms of monoamine oxidase (forms A and B) in rat brain, in studies with the drug inhibitor clorgyline (N-methyl-N-propargyl-3-(2,4dichlorophenoxy) -propylamine) : form A is very sensitive to clorgyline but form B is only inhibited by a high concentration of clorgyline. In contrast to clorgyline, the drug deprenyl (phenylisopropyhnethylpropinylamine) (11) inhibits form B at lower concentration than form A. Serotonin and norepinephrine are preferential substrates for form A monoamine oxidase and benxylamine and /3-phenylethylamine are prefer-
ential substrates for form B monoamine oxidase (12). Tyramine is oxidized by both forms (12). In the present study, using mixtures of two substrates, we compared the inhibitory and catalytic effects of various monoamine on forms A and B monoamine oxidase in mitochondrial preparations from rat brain. MATERIALS
Copyright 0 1978 by Academic Press, Inc. AU rights of reproduction in any form reserved.
METHODS
Male Wistar strain rate weighing 150 to 200 g were decapitated and their brains were removed and homogenized in 10 volumes of 0.32 M sucrose. The mitochondrial fraction was prepared by differential centrifugation using the method of Koenig et al. (13). The mitochondria were washed once by resuspending them in 0.32 M sucrose solution and used as the enzyme preparation. Monoamine oxidase activity was estimated essentially as described by Fuller (14) with [“C]tyramine and [‘%]serotonin and by Ekstedt (15) with [“Cltryptamine and P-[“C]phenylethylamine as substrates. The incubation medium contained various concentrations of radioactive substrates in a total volume of 275 d of potassium phosphate buffer, pH 7.2. The reaction was started by adding 25 al of the enzyme preparation. Incubation was carried out for 20 min at 37’C and then the reaction was stopped by adding 0.2 ml of 2 N HCl. With tyramine and serotonin, the reaction products were extracted with ether for 15 s in a homomixer and with tryptamine and /3-phenylethylamine 714
0003-9861/78/1912-0714$02.00/O
AND
MULTIPLE
CATALYTIC
SITES OF MONOAMINE
the products were extracted with toluene for 15 mm in a shaker. Samples of the extracts were mixed with 6 ml of Aquasol as scintillation liquid and their radioactivities were measured with a Packard Tri-Carb Liquid Scintillation Spectrometer. The enzyme activity was expressed in disintegrations per minute (dpm) extracted after 20 min of incubation. The dpm was a linear proportionality during the incubation time. Protein content was estimated by the method of Lowry et al. (16) and protein concentrations of the enzyme preparation were adjusted to 40 mg/ml. Mitochondriel treatments with clorgyline and deprenyl: Mitochondrial preparations were preincubated with either clorgyline (1 w) or deprenyl (10 pM) in a total volume of 1.0 ml at 37°C for 4 h (17), and then the monoamine oxidase activity of samples of the mixtures was estimated. Preparations treated with clorgyline had 98% of the control activity toward pphenylethylamine and 2% of that toward serotonin. Conversely, preparations treated with deprenyl had 2% and 60% of the control activities toward /3-phenylethylamine and serotonin, respectively. Thus, forms A and B were irreversibly inhibited by clorgyline and deprenyl(171, respectively, and in subsequent experiments, preparations treated with deprenyl and clorgyline were used as preparations of form A and B of monoamine oxidase, respectively. Deprenyl was kindly provided by Prof. Knoll, Budapest, Hungary and clorgyline from May & Baker Ltd., Degenham, England.
715
OXIDASE
showed lower K,,, values for tryptamine and P-phenylethylamine than for tyramine and serotonin. The K,,, values of the untreated preparation for tyramine, tryptamine, and /3-phenylethylamine obtained were the same as those of the form B enzyme, and the K,,, value for serotonin was the same as that of the form A enzyme.
Reaction Rates with a Mixture Substrates
of
Two
Figures 1 through 4 show the inhibitions by other substrates of the oxidations of labeled serotonin and P-phenylethylamine with untreated (Figs. 1 and 2), deprenyltreated (Fig. 3), and clorgyline-treated (Fig. 4) preparations. The reaction rate was es-
RESULTS
Determination of K,,, Values The K,,, values of various substrates for the untreated preparation (forms A and B) and the deprenyl-treated (form A) and clorgyline-treated (form B) preparations were calculated from Lineweaver-Burk plots. The results with serotonin, tyramine, tryptamine, and P-phenylethylamine as substrates are shown in Table I. The monoamine oxidase in rat brain
FIG. 1. Inhibition by various monoamines of serotonin oxidation by rat brain mitochondrial monoamine oxidase. Lineweaver-Burk plots of the reciprocal of the initial velocity of serotonin oxidation against the reciprocal of the labeled serotonin concentration in the presence of unlabeled monoamines at concentrations of twice their K, values. X-X, 30 pM tryptamine; A-A, 200 w tyramine; M, 4.6 JLM pphenylethylamine; M, no additional monoamine. The results are means of triplicate assays.
TABLE I K, VALUES FOR MONOAMINE OXIDASE IN UNTREATED, DEPRENYL-TREATED RAT BRAIN MITOCHONDRIA”
Serotonin Tyramine Tryptamine P-Phenylethylamine
Untreated mitochondria (PM) 89 100 15 2.3
Form A* deprenyl-treated mitochondria (PM) 80 51 5
AND CLORGYLINE-TREATED
Form B** clorgylme-treated mitochondria (PM) 120 15 1.6
a Bat brain mitochondria were preincubated with either deprenyl (10 F)* or clorgyline (1 PM)‘* in a total volume of 1.0 ml at 37°C for 4 h (see Materials and Methods) and then monoamine oxidase activity was estimated. K,,, values were obtained from Lineweaver-Burk plots with various concentrations of labeled substrates. The results are means of triplicate assays.
716
EGASHIRA,
KUROIWA,
AND
KAMIJO
I,,-PHE”ILETHILW,YE ‘ , innI-‘, FIG. 2. Inhibition
by various monoamines of pphenylethylamine oxidation of rat brain mitochondrial monoamine oxidase. Lineweaver-Burk plots .of the reciprocal of the initial velocity of /I-phenylethylamine oxidation against the reciprocal of the labeled P-phenylethylamine concentration in the presence of unlabeled monoamines at concentrations of twice their K,,, values. X-X, 30 PM tryptamine; A-A, 200 pM tyramine; M, 178 pM serotonin, u, no additional monoamine. The results are means of triplicate assays.
FIG. 4. Inhibition by various monoamines of pphenylethylamine oxidation by clorgylme-treated mitochondrial monoamine oxidase. Lineweaver-Burk plots of the reciprocal of the initial velocity of /3phenylethylamine oxidation against the reciprocal of the labeled P-phenylethylamine concentration in the presence of unlabeled monoamines at concentrations of twice their Km values. X-X, 30 PM tryptamine; A-A, 240 pM tyramine; o---O, 178 PM serotonm, u, no additional monoamine. The results are means of triplicate assays.
lines crossed at a single point on the ordinate, showing that tyramine and tryptamine are competitive inhibitors of serotonin oxidation. However, the plot of $ vs. 1
-10
IO I, SEROTOWI”
,
( .W’,
FIG. 3. Inhibition by various monoamines of serotonin oxidation by deprenyl-treated mitochondrial monoamine oxidase. Lineweaver-Burk plot of the reciprocal of the initial velocity of serotonin oxidation against the reciprocal of the labeled serotonin concentration in the presence of unlabeled monoamines at concentrations of twice their K,,, values. X-X, 10 PM tryptamine; A-A, 102 pi tryamine; M, 4.6 PM /3-phenylethylamine; M, no additional monoamine. The results are means of triplicate assays.
timated by measuring the radioactivity of the oxidation product. As shown in Figs. 1 and 3, with the untreated or form A preparation, values of i 1 gave straight lines in the [ serotonin] presence and absence of tyramine, tryptamine, and /3-phenylethylamine. These us
in the presence of P-phenyleth[serotonin] ylamine coincided with the control line, indicating that P-phenylethylamine does not bind with form A enzyme or inhibit the exidation of serotonin. These results are understandable since forms A and B do not react with /3-phenylethylamine and serotonin, respectively. Figures 2 and 4 show plots of the reciprocals of the rates of the reaction and of pphenylethylamine concentration for untreated and form B preparations in the presence and absence of tyramine, tryptamine, and serotonin. The data indicate that tyramine and tryptamine are competitive inhibitors of P-phenylethylamine oxidation. Although serotonin was not oxidized by the form B enzyme, the data show that serotonin is a competitive inhibitor of P-phenylethylamine oxidation. Therefore, serotonin seems to bind to the active site of the form B as an inhibitor. DISCUSSION
The Km values of rat brain mitochondrial monoamine oxidase were calculated from
MULTIPLE
CATALYTIC
SITES
OF MONOAMINE
717
OXIDASE
amine oxidase reaction:
Lineweaver-Burk plots with four labeled substrates. P-Phenylethylamine had the lowest Km value for untreated mitochondria and form B monoamine oxidase. The Km value of the untreated preparations for serotonin was the same as that of the form A enzyme, whereas the Km values for tyramine, tryptamine, and /I-phenylethylamine were similar to those of the form B enzyme. The differences in the K,,, values of the forms A and B for tyramine and tryptamine were comparable. These findings suggest that these substrates are oxidized by both forms of mitochondrial monoamine oxidase, and that when untreated mitochondria are used as an enzyme source, tyramine, tryptamine, and j%phenylethylamine are oxidized predominantly by the form B, although the form A has a higher affinity for these substrates. We measured the oxidation rates of various substrates in the presence of another substrate using untreated mitochondria, and the forms A and B of enzyme from rat brain, because if two substrates are oxidized at the same site, the presence of the one will competitively inhibit oxidation of the other, whereas if they are oxidized by different enzymes, the presence of one should not affect oxidation of the other. When there are two substrates, S1 and Sp, in the reaction mixture, the following mechanism may be proposed for the mono-
KAl EA+ SI +EA&
K1 -EA
+ PI
PI
+ Pz
PI
+ P,
[31
K2’ EeSz Ee + Pz
[41
EA+SZ- KAZ\E&-E+,
K,’
I%+.‘%-xEeSl-EB EB+&-
Ke,,
K2
where EA and Es are the form A and B enzymes, KA,, KAY, KB~, and KB, are the Michaelis constants of the respective reactions and PI and Pz the products. When S1 is serotonin and SP is tyramine or tryptamine, the reaction 3 will not occur, because the form B does not oxidize serotonin. The rate v1 of oxidation of S1 can be expressed as follows:
--=Wll
v1 -
WI1
PI
dt
[Sl] + KAY
When S1 is /3-phenylethylamine and SZ is tyramine or tryptamine, the following equation can be obtained from equations 3 and 4, V’[Sl j Vl = (6) [Sl] + Kg,
TABLE
II
VALUESOF KAp AND KBZ” Enzyme
S*
SP
KAY or I&
&G Untreated Mitochondria (Forms A and B)
Deprenyl-treated chondria (Form A) Clorgyline-treated chondria (Form B)
SERO
89
PEA
2.3
mito-
SERO
80
mito-
PEA
1.6
TYRA TRYPT PEA TYRA TRYPT SERO TYRA TRYPT PEA TYRA TRYPT SERO
100 15 2.3 106 15 89 51 5 2.3 120 15 89
104 7 155 14 320 129 5 113 10 480
’ Rat brain untreated, deprenyl-treated, and clorgyline-treated mitochondria were used as enzyme sources. The concentration of labeled substrates, S, was varied in the presence or absence of another substrate, Sp, at a fiied concentration of twice the K,,, value. K,,, values are quoted from Table I. The apparent Michaelis constants in the presence of Sz were determined from the slopes of double reciprocal plots using the data in Figs. 1-4. SERO, serotonin; TYRA; tyramine; TRYPT, tryptamine; PEA, /I-phenylethylamine. The values are means of triplicate assays.
718
EGASHIRA,KUROIWA,
AND
The KA, and Kg, values were calculated from annarent Michaelis constants, KA, (1 +‘e)
and KB~( 1 +e)
using
KAMIJO REFERENCES
1. YANG, H. Y. T., GORIDIS, C., AND NEFF, N. H. (1972) J. Neurochem. 19, 1241-1250. 2. YANG, H. Y. T., AND NEFF, N. H. (1973) J. Pharmacol. Exp. Ther. 187,365-371. 3. FULLER, R. W. (1972) Adu. Biochem. Psycho-
the data sho& in Figs. l-4. These values obtained (Table II) were similar to those in Table I calculated from Lineweaver-Burk pharmacol. 5,339-354. plots of values in the presence of only one YABUHARA, H. (1974) Japan. J. Pharmacol. 24, substrate. Form B did not oxidize serotonin. 523-533. /&Phenylethylamine did not inhibit the oxMCCAULEY, R., AND RACKER, E. (1973) Mol. Cell. Biochem. 1, 73-81. idation of serotonin, but serotonin inhibited YOUDIM, M. B. H. (1974) Adu. Biochem. Psychothe oxidation of P-phenylethylamine by the pharmacol. l&59-63. untreated preparation or the form B of 7. COLLINS, G. G. S., SANDLER, M., WILLIAMS, E. D., enzyme. AND YOUDIM, M. B. H. (1970) Nature 225, The K,,, value for serotinin was 80-89 817-820. PM with a single substrate, while the Kg, 8. SHIH, J., AND EIDUSON, S. (1971) J. Neurochem. value of this substrate was 320-480 pM (see 18, 1221-1227. Table II). These results show that serotonin 9. JAIN, M. L., AND SANDS, F. L. (1974) J. Neurocan bind to form B enzyme with very weak them. 23, 1291-1293. affinity and not be oxidized by it. 10. JOHNSTON, J. P. (1966) B&hem. Pharmacol. 17, 1285-1297. Tryptamine has been identified in human 11. KNOLL, J., AND MAGYAR, K. (1972) Adu. Biochem. urine (18) and certain tissues, including Psychopharmacol. 5,393-407. brain (19, 20), and it has been reported to be related to etiological factors of schizo- 12. NEFF, N. H., AND YANG, H. Y. T. (1974) Life Sci. 14,2061-2074. phrenia (21), depression (22) and parkin- 13. KOENIG, H., GAINES, D., MCDONALD, T., GRAY, sonism-like symptoms (23). Moreover, R., AND SCOTT, J. (1964) J. Neurochem. 11, tryptamine was recently found in the cen729-740. tral nervous system, suggesting that it may 14. FULLER, R. W. (1968) Arch. Int. Pharmacodyn. act at the same receptor sites as serotonin 174.32-36. in the brain (24). It has also been found in 15. EKSTEDT, B. (1976) Biochem. Pharmacol. 25, 1133-1138. both serotonergic and catecholaminergic 16. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., neurons, suggesting its transmitter function (25).
The comparison between tryptamine and serotonin would presume the strong inhibitory action of tryptamine on serotonin oxidation by untreated mitochondria and form A enzyme from rat brain, and thus it may regulate the metabolism of serotonin. However, tryptamine also strongly inhibits P-phenylethylamine oxidation. Thus, besides binding to forms A and B, tryptamine is a substrate for both forms in the brain. Therefore, studies on the concentration of tryptamine may serve to elucidate the functional significance of monoamine oxidase in the brain. ACKNOWLEDGMENT We are sincerely grateful to Profeeeor Yasuyuki Ogura (Department of Biochemistry, Tchci University) for his helpful discussion and advice.
17. 18. 19.
20. 21.
AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. EGASHIRA, T., EKSTEDT, B., AND ORELAND, L. (1976) B&hem. Pharmacol. 25,2583-2586. PERRY, T. L., AND SCHROEDER, W. A. (1963) J. Chromatogr. 12,368-372. MARTIN, W. R., SLOAN, J. W., CHRISTIAN, S. T., AND CLEMENTS, T. H. (1972) Psychopharmacologia 24,331-346. SAAVEDRA, J. H., AND AXELROD, J. (1972) J. Pharmacol. Exp. Ther. 182,363-369. BRUNE, G. G., AND HIMWICH, H. E. (1962) Arch.
Gen. Psychiatr. 6, 324-346. 22. DEWHURST, W. G. (1968) Nature 218,1130-1133. 23. SAAVEDRA, J. M., HELLER, B., AND FISCHER, E. (1970) Nature 220,868. 24. TEDESCHI, D. H., TEDESCHI, R. E., AND FELLOWS, E. J. (1959) J. PhurntacoZ. Exp. Ther. 126, 223-231. 25. MARSDEN, C. A., AND CURZON, G. (1974) J. Neurochem. 23, 1171-1176.
