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A radiochemical method for monoamine oxidase assay
ANALYTICAL
BIOCHEMISTRY
A Radiochemical
30, 3‘i@-i+i5 (1969)
Method
J. H. FELLMAN, Department
for Monoamine
E. S. ROTH,
of Biochemistry Medical
Oxidase
Assay’
AND R. F. MOLLICA
and Division of NeuroLogy, University School, Portland, Oregon 97201
of Oregon
Received November 20, 1968 The interest in the catecholamines as neurohumoral agents has encouraged the development of sensitive assays for one of the major enzymes responsible for their catabolism, monoamine oxidase (EC 1.4.3.4, monoamine:oxygen oxidoreductase (deaminating) ) . A number of chemical methods are available for the assay of monoamine oxidase activity (l-3). These have been more recently supplemented with highly sensitive radiochemical procedures using 14C amines as substrates (4, 5). In the course of our investigations on the metabolism of tyrosine, we developed an assay method for tyrosine aminotransferase based upon the release of tritium from the 2,3 side chain position of tyrosine (6). We report here an extension of this principle and describe a method for the assay of monoamine oxidase activity. This method depends upon the release of tritium from the 1,2 positions of tyramine when it is oxidized to p-hydroxyphenylacetaldehyde by monoamine oxidase, and subsequently to p-hydroxyphenylacetic acid by aldehyde oxidase. The low cost of the tritiated tyramine and the relative low quenching achieved after separation of the tritiated water from the substrate make this a useful procedure for routine assay and monoamine oxidase inhibitor studies. METHODS 1. Preparation of tyramine-1,2-3H by decarboxylution of L-tyrosine2,S3H. n-Tyrosine (900 mg) was mixed with 500 ,UC of n-tyrosine-2,33H (500 J.L~of a 5 mc/5 ml solution of this substance, obtained from Nuclear-Chicago with a specific activity of 3.57 curies/mmole) and brought to dryness under vacuum. Diphenylamine (8 gm) was added and the mixture slowly heated to 240”. At this temperature one can observe the appearance of bubbles of COZ. Heating was continued for 30 min until all of the tyrosine dissolved in the diphenylamine melt and all %O, evolution ceased. The tube was cooled and extracted with 50 ml ‘This work was supported by U. S. Public Health Service Grant NB-1572 the National Institute of Neurological Diseases and Blindness. 339
from
340
FELLMAN,
ROTH,
AND
MOLLICA
of dry ethyl ether. The fluffy residue was dissolved in ethanol, filtered to remove any insoluble materials, and acidified with 10 ml of 3 N HCl. The solution was evaporated to dryness in a rotary evaporator. The residue was recrystallized from ethanol to yield 360 mg (40%) of colorless needles of tyramine hydrochloride. Radiochemical assay showed that all of the tritium was recovered and that no exchange of tritium had occurred. The final specific activity of the recrystallized product was 100 JLC3H/mmole. 2. Enzyme source. A number of enzyme sources was used, but for all of the results given below dialyzed rat liver homogenates were employed. Male Sprague-Dawley rats were sacrificed and their livers homogenized in 0.1 M phosphate buffer, pH 7.8, and dialyzed against the same buffer. 3. Assay procedure. To a solution containing 1 ml of phosphate buffer, pH 7.8, was added 1 ml of a solution of tritiated tyramine, 4 X 1O-3 M (100 &mmole) (0.4 ye/reaction vessel), prepared as described above. I600-
1400
-
1200-
IOOO4 .:: -s
soo-
G 600 -
I
I
I
I
I
I
5
6
7
8
9
IO
PH FIG. 1. Effect of pH on velocity of monoamine oxidase activity. Phosphate buffers (0.1 M) were employed to maintain pH indicated. Activity values represent cpm 8H/30 min/ml liver homogenate as described in assay procedure.
MONOAMINE
OXIDASE
341
ASSAY
At zero time, 1 ml of enzyme homogenate containing 6 mg protein per milliliter was added; a control vessel contained 1 ml of boiled enzyme homogenate. Under the conditions employed the boiled enzyme control gave 10 cpm above background. The reaction tubes were incubated at 37” with shaking for varying periods. Usually 30 min was employed for assay. At the end of the incubation period the tubes were plunged into boiling water and centrifuged and the supernatant poured over charcoal columns. These were placed in 1.5 x 10 cm polyethylene tubes and centrifuged at 1000 rpm for 5 min to obtain a clear filtrate, free of tyramine and p-hydroxyphenylacetaldehyde. In some instances the filtrate was microdistilled. In these cases all of the radioactivity was recovered as tritiated water, indicating the thorough separation of the radioactive substrate from the radioactive water. Charcoal columns were made by placing 500 mg of activated charcoal (Nuchar C-190N from Matheson, Coleman and Bell) in a 0.5 X 12 cm glass column fitted with a sintered glass disc. The columns were packed dry by centrifuging at 1000 rpm for 5 min.
CPM
20
40 Time
60
SO
(min)
FIG. 2. Rate of release of tritium from tyramine-1,2-*H by monoamine oxidase. 1 ml dialyzed liver homogenate containing 6 mg protein was used as enzyme source.
342
FELLMAN,
ROTH,
AND
MOLLICA
Release of tritium was determined by placing 0.5 ml of filtrate and 19 ml of a solution containing 2 gm of PPO, 50 mg of POPOP in 250 ml of absolute ethanol plus 250 ml of toluene (7) in a scintillation vial. The samples were counted with a Beckman CPM 100 liquid scintillation counter. RESULTS
AND
DISCUSSION
A number of parameters was studied to characterize the enzyme activity using the procedure described above. The effect of pH on the rate of the reaction is given in Figure 1. These data compare favorably with the results of Malafaya-Baptista et al. (8). The oxidation of tyramine was initially linear with respect to time but as shown in Figure 2 the rate slowly decreased. The rate of the reaction was linearly related to the amount of enzyme used in the assay, as shown in Figure 3. Some deviation from linearity was noted at the higher enzyme levels employed. The effect of substrate on the velocity of the reaction was studied and the data plotted according to the method of Lineweaver and Burk (Fig. 4). We observed a Km value of 3.3 X 1O-4 mole/liter, determined by either measuring the velocity of the over-all reaction or trapping the aldehyde formed with semicarbazide and thus measuring the rate of the monoamine oxidase activity. This value differed from that of Davidson (9)) who reported a Km value of 3 X 1O-3 mole/liter using oxygen uptake
0.2
0.4
O$
0.8
1.0
ml FIG. 3. Relationship of rat& of reaction to amount of enzyme used. 1 ml homogenate solution conkained 6 mg protein. Monoamine oxidase &ctiVity is expressed in > iv.:- : cpm/36 min incubation at 37”.
MONOAMINE
OXIDASE
343
ASSAY
6
2 &LE I/ /’
0
I
1
i
f
I
4000
2000
J
6000
I/S
FIG. 4. Effect of substrate on enzyme velocity expressed by Lineweaver-Burl; method. Open circles, line obtained using 1 X 10m3M semicarbazide to inhibit oxidation of aldehyde product, Filled circles, data obtained in absence of semicarbazidc. Velocity of the reaction was measured as described in text.
to study monoamine oxidase activity. Green and Haugton (1)) however, reported Km values of 3.0 X 1e4 mole/liter and (10) 2.2 X 10m4 mole/ liter, and Oswald and Strittmatter (11) 8.4 X 1O-4 mole/liter using tyramine as substrate. Higher substrate concentrations were inhibitory and perhaps may be a factor in the variation of Km values observed among various investigators. The two radiochemical procedures using tryptamine-W (4) and tyramineJ4C (5) employ substrate concentrations in the order of 2 X IO-” and 1.3 X 1O-5 mole/liter, respectively. Employing these low substrate concentrations could lead to error in assay since they approach the limiting substrate concentrations to obtain zero-order kinetic conditions. The effect of a-methylphenylethylhydrazine (Cantron2), an inhibitor of ’ Catron, manufactured
by Lakeside Laboratories
Inc., Milwaukee,
Wise.
FELLMAN,
ROTH,
AND
MOLLICA
Concentration FIG. 5. Inhibitory effect of cu-methylphenylethylhydrazine of monoamine oxidase.
(Catron)
on activity
monoamine oxidase, was studied. The inhibitory effect of this substance is related to the log of the concentration of inhibitor (Fig. 5) and illustrates the application of the method for the study of monoamine oxidase inhibitors. SUMMARY
A simple radiochemical method is described for the assay of monoamine oxidase utilizing tritiated tyramine. The synthesis of tyraminel,2-3H from tyrosine-2,3-3H is reported. Tritium, released by the oxidation of tyramine-l,2-3H by monoamine oxidase, was counted by conventional liquid scintillation techniques. Also reported is the application of this method for the study of a number of parameters which influence the activity of the enzyme, including pH, substrate concentration, and amount of enzyme. REFERENCES GREEN, A. L., AND HATJGTON, T. M., Biochem. J. 78, 172 (1961). COTZIAS, G. C., AND DOLE, V. P., J. Biol. Chem. 190, 665 (1951). CREASEY, N. H., Biochem. J. 64, 178 (1956). WURTMAN, R. J., AND AXELROD, J., Biochem. Pharmac. 12, 1439 (1963). OTSUKA, S., AND KOBAYASHI, Y., Biochem. Pharmac. 13, 995 (1964). FELLMAN, J. H., VANBELLINGHEN, P. J., KOLER, R. D., ANB JONES, R. Biochemktry 8, 615 (1969). 7. BUHLER, D. R., Anal. Biochem. 4, 413 (1962). 8. MALAFAYA-BAPTISTA, A., GARRETT, J., OSSWALD, W., AND MALAFAYA-BAPTISTA, F., Arch. Exptl. Pathol. Pharmakol. 230, 10 (1957). 1. 2. 3. 4. 5. 6.
T.,
M.
MONOAMINE
OXIDASE
ASSAY
9. DAVIDSON, A. N., Btichm. J. 67, 316 (1957). 10. GRFBN, A. L., Biochem. Phammc. 13, 249 (1964). 11. OSWALD, E. O., AND STRITTMAITER, C. F., hoc. Sot. Exptl. (1963).
345
Biol. Ned. 114, 66s
BIOCHEMISTRY
A Radiochemical
30, 3‘i@-i+i5 (1969)
Method
J. H. FELLMAN, Department
for Monoamine
E. S. ROTH,
of Biochemistry Medical
Oxidase
Assay’
AND R. F. MOLLICA
and Division of NeuroLogy, University School, Portland, Oregon 97201
of Oregon
Received November 20, 1968 The interest in the catecholamines as neurohumoral agents has encouraged the development of sensitive assays for one of the major enzymes responsible for their catabolism, monoamine oxidase (EC 1.4.3.4, monoamine:oxygen oxidoreductase (deaminating) ) . A number of chemical methods are available for the assay of monoamine oxidase activity (l-3). These have been more recently supplemented with highly sensitive radiochemical procedures using 14C amines as substrates (4, 5). In the course of our investigations on the metabolism of tyrosine, we developed an assay method for tyrosine aminotransferase based upon the release of tritium from the 2,3 side chain position of tyrosine (6). We report here an extension of this principle and describe a method for the assay of monoamine oxidase activity. This method depends upon the release of tritium from the 1,2 positions of tyramine when it is oxidized to p-hydroxyphenylacetaldehyde by monoamine oxidase, and subsequently to p-hydroxyphenylacetic acid by aldehyde oxidase. The low cost of the tritiated tyramine and the relative low quenching achieved after separation of the tritiated water from the substrate make this a useful procedure for routine assay and monoamine oxidase inhibitor studies. METHODS 1. Preparation of tyramine-1,2-3H by decarboxylution of L-tyrosine2,S3H. n-Tyrosine (900 mg) was mixed with 500 ,UC of n-tyrosine-2,33H (500 J.L~of a 5 mc/5 ml solution of this substance, obtained from Nuclear-Chicago with a specific activity of 3.57 curies/mmole) and brought to dryness under vacuum. Diphenylamine (8 gm) was added and the mixture slowly heated to 240”. At this temperature one can observe the appearance of bubbles of COZ. Heating was continued for 30 min until all of the tyrosine dissolved in the diphenylamine melt and all %O, evolution ceased. The tube was cooled and extracted with 50 ml ‘This work was supported by U. S. Public Health Service Grant NB-1572 the National Institute of Neurological Diseases and Blindness. 339
from
340
FELLMAN,
ROTH,
AND
MOLLICA
of dry ethyl ether. The fluffy residue was dissolved in ethanol, filtered to remove any insoluble materials, and acidified with 10 ml of 3 N HCl. The solution was evaporated to dryness in a rotary evaporator. The residue was recrystallized from ethanol to yield 360 mg (40%) of colorless needles of tyramine hydrochloride. Radiochemical assay showed that all of the tritium was recovered and that no exchange of tritium had occurred. The final specific activity of the recrystallized product was 100 JLC3H/mmole. 2. Enzyme source. A number of enzyme sources was used, but for all of the results given below dialyzed rat liver homogenates were employed. Male Sprague-Dawley rats were sacrificed and their livers homogenized in 0.1 M phosphate buffer, pH 7.8, and dialyzed against the same buffer. 3. Assay procedure. To a solution containing 1 ml of phosphate buffer, pH 7.8, was added 1 ml of a solution of tritiated tyramine, 4 X 1O-3 M (100 &mmole) (0.4 ye/reaction vessel), prepared as described above. I600-
1400
-
1200-
IOOO4 .:: -s
soo-
G 600 -
I
I
I
I
I
I
5
6
7
8
9
IO
PH FIG. 1. Effect of pH on velocity of monoamine oxidase activity. Phosphate buffers (0.1 M) were employed to maintain pH indicated. Activity values represent cpm 8H/30 min/ml liver homogenate as described in assay procedure.
MONOAMINE
OXIDASE
341
ASSAY
At zero time, 1 ml of enzyme homogenate containing 6 mg protein per milliliter was added; a control vessel contained 1 ml of boiled enzyme homogenate. Under the conditions employed the boiled enzyme control gave 10 cpm above background. The reaction tubes were incubated at 37” with shaking for varying periods. Usually 30 min was employed for assay. At the end of the incubation period the tubes were plunged into boiling water and centrifuged and the supernatant poured over charcoal columns. These were placed in 1.5 x 10 cm polyethylene tubes and centrifuged at 1000 rpm for 5 min to obtain a clear filtrate, free of tyramine and p-hydroxyphenylacetaldehyde. In some instances the filtrate was microdistilled. In these cases all of the radioactivity was recovered as tritiated water, indicating the thorough separation of the radioactive substrate from the radioactive water. Charcoal columns were made by placing 500 mg of activated charcoal (Nuchar C-190N from Matheson, Coleman and Bell) in a 0.5 X 12 cm glass column fitted with a sintered glass disc. The columns were packed dry by centrifuging at 1000 rpm for 5 min.
CPM
20
40 Time
60
SO
(min)
FIG. 2. Rate of release of tritium from tyramine-1,2-*H by monoamine oxidase. 1 ml dialyzed liver homogenate containing 6 mg protein was used as enzyme source.
342
FELLMAN,
ROTH,
AND
MOLLICA
Release of tritium was determined by placing 0.5 ml of filtrate and 19 ml of a solution containing 2 gm of PPO, 50 mg of POPOP in 250 ml of absolute ethanol plus 250 ml of toluene (7) in a scintillation vial. The samples were counted with a Beckman CPM 100 liquid scintillation counter. RESULTS
AND
DISCUSSION
A number of parameters was studied to characterize the enzyme activity using the procedure described above. The effect of pH on the rate of the reaction is given in Figure 1. These data compare favorably with the results of Malafaya-Baptista et al. (8). The oxidation of tyramine was initially linear with respect to time but as shown in Figure 2 the rate slowly decreased. The rate of the reaction was linearly related to the amount of enzyme used in the assay, as shown in Figure 3. Some deviation from linearity was noted at the higher enzyme levels employed. The effect of substrate on the velocity of the reaction was studied and the data plotted according to the method of Lineweaver and Burk (Fig. 4). We observed a Km value of 3.3 X 1O-4 mole/liter, determined by either measuring the velocity of the over-all reaction or trapping the aldehyde formed with semicarbazide and thus measuring the rate of the monoamine oxidase activity. This value differed from that of Davidson (9)) who reported a Km value of 3 X 1O-3 mole/liter using oxygen uptake
0.2
0.4
O$
0.8
1.0
ml FIG. 3. Relationship of rat& of reaction to amount of enzyme used. 1 ml homogenate solution conkained 6 mg protein. Monoamine oxidase &ctiVity is expressed in > iv.:- : cpm/36 min incubation at 37”.
MONOAMINE
OXIDASE
343
ASSAY
6
2 &LE I/ /’
0
I
1
i
f
I
4000
2000
J
6000
I/S
FIG. 4. Effect of substrate on enzyme velocity expressed by Lineweaver-Burl; method. Open circles, line obtained using 1 X 10m3M semicarbazide to inhibit oxidation of aldehyde product, Filled circles, data obtained in absence of semicarbazidc. Velocity of the reaction was measured as described in text.
to study monoamine oxidase activity. Green and Haugton (1)) however, reported Km values of 3.0 X 1e4 mole/liter and (10) 2.2 X 10m4 mole/ liter, and Oswald and Strittmatter (11) 8.4 X 1O-4 mole/liter using tyramine as substrate. Higher substrate concentrations were inhibitory and perhaps may be a factor in the variation of Km values observed among various investigators. The two radiochemical procedures using tryptamine-W (4) and tyramineJ4C (5) employ substrate concentrations in the order of 2 X IO-” and 1.3 X 1O-5 mole/liter, respectively. Employing these low substrate concentrations could lead to error in assay since they approach the limiting substrate concentrations to obtain zero-order kinetic conditions. The effect of a-methylphenylethylhydrazine (Cantron2), an inhibitor of ’ Catron, manufactured
by Lakeside Laboratories
Inc., Milwaukee,
Wise.
FELLMAN,
ROTH,
AND
MOLLICA
Concentration FIG. 5. Inhibitory effect of cu-methylphenylethylhydrazine of monoamine oxidase.
(Catron)
on activity
monoamine oxidase, was studied. The inhibitory effect of this substance is related to the log of the concentration of inhibitor (Fig. 5) and illustrates the application of the method for the study of monoamine oxidase inhibitors. SUMMARY
A simple radiochemical method is described for the assay of monoamine oxidase utilizing tritiated tyramine. The synthesis of tyraminel,2-3H from tyrosine-2,3-3H is reported. Tritium, released by the oxidation of tyramine-l,2-3H by monoamine oxidase, was counted by conventional liquid scintillation techniques. Also reported is the application of this method for the study of a number of parameters which influence the activity of the enzyme, including pH, substrate concentration, and amount of enzyme. REFERENCES GREEN, A. L., AND HATJGTON, T. M., Biochem. J. 78, 172 (1961). COTZIAS, G. C., AND DOLE, V. P., J. Biol. Chem. 190, 665 (1951). CREASEY, N. H., Biochem. J. 64, 178 (1956). WURTMAN, R. J., AND AXELROD, J., Biochem. Pharmac. 12, 1439 (1963). OTSUKA, S., AND KOBAYASHI, Y., Biochem. Pharmac. 13, 995 (1964). FELLMAN, J. H., VANBELLINGHEN, P. J., KOLER, R. D., ANB JONES, R. Biochemktry 8, 615 (1969). 7. BUHLER, D. R., Anal. Biochem. 4, 413 (1962). 8. MALAFAYA-BAPTISTA, A., GARRETT, J., OSSWALD, W., AND MALAFAYA-BAPTISTA, F., Arch. Exptl. Pathol. Pharmakol. 230, 10 (1957). 1. 2. 3. 4. 5. 6.
T.,
M.
MONOAMINE
OXIDASE
ASSAY
9. DAVIDSON, A. N., Btichm. J. 67, 316 (1957). 10. GRFBN, A. L., Biochem. Phammc. 13, 249 (1964). 11. OSWALD, E. O., AND STRITTMAITER, C. F., hoc. Sot. Exptl. (1963).
345
Biol. Ned. 114, 66s