BIOCHEMICAL
MEDICINE
18, 378-383
(1977)
Effect of Oxygen on Human Platelet Monoamine Oxidase KIYOFUMI Neuropsychiatric Biobehavioral
KOBAYASHI'
AND SAMUEL EIDUSON
and Brain Research Institutes, Departments Sciences and Biological Chemistry, UCLA University of California at Los Angeles, Los Angeles, California 90024
of Psychiatry and Medical School,
Received April 5, 1977
The enzyme monoamine oxidase (MAO; E.C. 1.4.3.4.) is involved in the oxidative deamination of physiologically important monoamines (dopamine, norepinephrine, serotonin, and other biogenic amines) and is widely distributed in human tissues. Human blood platelets have been found to contain MAO (l), and a reduced MAO activity in platelets has recently been proposed as a genetic marker for vulnerability to schizophrenia (2-4). Although it has generally been accepted that MAO in other tissues such as brain and liver exists in multiple forms, this has not been thought to be the case for platelet MAO (5, 6). However, it has recently been reported that human platelet MAO contained multiple catalytic sites (6, 7). Furthermore, it has beeen observed in our laboratory, using a variety of techniques (8), that there may be at least two distinct conformations of human platelet MAO. It has also been reported that MAO activity in the liver, thyroid, heart, and brain of various animals was affected by the oxygen tension of the assay environment (9-l 1). More recently, Roth (12) has compellingly suggested that, at levels of oxygen of about 0.9 mM, phenethylamine and benzylamine “. . . share a common catalytic site . . .” and also bind to the reduced form of the enzyme. The present paper describes a study of the effect of oxygen on human platelet MAO using three different substrates at three different incubation temperatures and provides evidence that the catalytic site(s) of the human platelet enzyme may respond differentially to various substrates at different temperatures under these conditions. I Present address: Institute for Neurobiology, 2-5-l Shikata-cho, Okayama, Japan.
Okayama University Medical School,
378 Copyright All rights
@ 1977 by Academic Press, Inc. of reproduction in any form recerved
ISSN 0006-2944
OXYGEN
AND
HUMAN
PLATELET
MONOAMINE
OXIDASE
379
MATERIALS AND METHODS Human platelets were isolated from blood collected in citrate as described previously (13) and were stored in a freezer at -20°C. The isolated platelets were solubilized by sonication of the platelets for 5 set before assay. MAO activity was assayed radiochemically with three different substrates [side chain-2J4Cltryptamine bisuccinate and /3-El-L4Clphenylethylamine hydrochloride, New England Nuclear; [methylene J4C]benzylamine hydrochloride, ICN Pharmaceuticals, Inc.) and at three different temperatures (20, 37, and 42°C). Unlabeled substrates were obtained as follows: tryptamine hydrochloride and benzylamine from Sigma Chemical Co., /3-phenylethylamine from Eastman Kodak Co. The final concentrations of substrates were 1 x 10-5~. The enzyme preparation (in 0.5 M phosphate buffer, pH 7.4) was incubated at the appropriate temperature for 20 min. The reaction was then initiated by the addition of the substrate, and incubation was carried out by shaking for 5 min in air or by bubbling 100% oxygen into the solution for 5 set just after adding substrate. The oxygenated reaction tube was stoppered and shaken for 5 min. The reaction was stopped with the addition of 0.1 ml of 6 N HCl. The products were extracted with 6 ml of toluene. Protein determination was carried out by the method of Lowry et al. (14). Statistical significance was determined using the Mann-Whitney U test. RESULTS The effect of oxygen on human platelet MAO activity at three different temperatures when assayed with the substrates benzylamine, tryptamine, and phenylethylamine is summarized in Table 1. Using phenylethylamine as substrate, MAO activity significantly increased with oxygen at all three temperatures. MAO activity also increased with oxygen at 20 and 37°C when assayed with tryptamine as substrate. On the other hand, when benzylamine was used as substrate no alteration of enzyme activity with oxygen was observed at any temperature (see also Fig. 1). Figure 1 shows the ratio of MAO activity obtained in oxygen to that in air. The ratios of MAO activity when assayed with phenylethylamine were 1.90, 2.16, and 2.42 at 20, 37, and 42”C, respectively. (These values were significantly higher (P < 0.01) than those obtained with tryptamine and benzylamine.) The ratios of MAO activity with tryptamine as substrate were significantly higher (P < 0.01) than those for benzylamine at 20 and 37”C, but not at 42°C. Unlike that observed with phenylethylamine, increased temperature significantly decreased the oxygen effect when using tryptamine as substrate. The ratios with benzylamine, however, were not significantly different at three temperatures.
(12)
(12)
(12)
0.173 + 0.016 (17) 0.224 5 0.019
(12)
0.058 -e 0.003
Air
(12)
0.228 t 0.021* (17) 0.252 + 0.023
(12)
0.089 2 0.006**
Tryptamine
(12)
1.244 2 0.100 (17) 1.306 k 0.129
(12)
0.597 k 0.053
Air
1.142 + 0.123*” (12) 2.736 _f 0.272” ’ (17) 3.134 z 0.2x1-: (12)
Oxygen
_____
Phenylethylamine
TEMPERATURES”
of protein/5 min)
AT THREE
Oxygen
SUBSTRATES
“Data given are mean values t SEM, with the number of cases in parentheses. Significance of the difference of MAO activity in oxygen as compared to air: *P < 0.05, **P < 0.001 (Mann-Whitney U test).
42
0.245 2 0.023 (17) 0.322 2 0.029
(12)
(12)
0.245 + 0.023 (17) 0.329 k 0.029
0.093 + 0.007
0.087 k 0.009
20
37
1
THREE
TABLE USING
MAO activity @mole oxidizedimg
MAO ACTIVITY
Oxygen
ON
Air
Benzylamine
OF OXYGEN
Incubation temperature (“C)
EFFECT
OXYGEN
AND HUMAN
PLATELET
MONOAMINE
OXIDASE
381
%r-+-i+ temperature
(*C
1
FIG. I. The ratio of monoamine oxidase activity obtained in oxygen to that in air when assayed with the substrates benzylamine ( C---m), tryptamine (LO), and phenylethylamine (&A) at three temperatures. Each point represents the mean values k SEM, calculated from the ratio in individual cases (number of cases are indicated in Table 1).
DISCUSSION
It has been reported that increased oxygen tension of the assay environment increased the activity of MAO from various tissues such as thyroid, liver, heart, and brain (9-12). However, the rate of increase of MAO activity by oxygenation varies with tissues, species, and substrates used at 37°C (12). In the present study, we found that the platelet MAO activity increased with oxygenation of the reaction mixture when using phenylethylamine (at three temperatures) and tryptamine (20 and 37°C) as substrate, while there were no alterations in the activity when using benzylamine as substrate. The rates of increase were about 100% with phenylethylamine and 30% with tryptamine at 37°C. Furthermore, the increase of MAO activity varied with the temperature, both with phenylethylamine and tryptamine. Although human platelet MAO is electrophoretically homogenous (5, 6), it appears to have multiple binding or catalytic sites (6,7) as does brain mitochondrial MAO (15,16). Edwards and Chang (6) reported that human platelet MAO contained two interacting catalytic sites on the basis of
382
KOBAYASHJ
AND EJDUSON
substrate competition
experiments; i.e., one active site had a high affinity the other had a high affinity for benzylamine and tryptamine. On the other hand, Roth (13) suggested that both PEA and benzylamine occupy the same site on the B form of the enzyme derived from brain tissue. In our preliminary studies using both ESR and “Clabeled substrates, human platelet enzyme appears to exist in at least two forms or to have two different binding sites (8). Our present results are consistent with the existence of at least two catalytic or binding sites in the human platelet enzyme. While recent studies suggested that platelet MAO activity was reduced in schizophrenics and may be a genetic marker for vulnerability to schizophrenia (2-4), opposite results have also been reported by other workers (17). The reason for such contradictory results is not clear. However, if human platelet MAO has one or more binding sites as indicated above and which are affected by oxygen concentration, the possibility should be raised that it may not be appropriate to relate total MAO activity in platelets to behavior disorders by determining MAO activity using only one substrate in air and at one temperature (37°C). What may be more significant than total enzyme activity in regard to such disorders is the nature of the catalytic or binding sites of the enzyme. for phenylethylamine,
SUMMARY Effect of oxygen on the determination of human platelet monoamine oxidase (MAO) was studied by using three different substrates at three different temperatures. MAO activity using phenylethylamine as substrate increased about two times with oxygenation at all three temperatures. On the other hand, there was virtually no alteration in activity using benzylamine as substrate at any temperature. Using tryptamine as substrate, the rate of increase in MAO activity varied with the temperature used. Our findings suggest that the catalytic site(s) of the human platelet enzyme responds differentially to various substrates at different temperatures under different concentrations of oxygen. ACKNOWLEDGMENTS This work was supported by the Ralph E. Smith Foundation and National Institute of Mental Health Grant 19734-06. We are grateful to Ms. Ruth Chang for her skillful assistance.
REFERENCES 1. Paasonen, M. K., Solatunturi, E., and Kivalo, E., Psychopharmacologia 6, 120 (1964). 2. Murphy, D. L., and Wyatt, R., Nature (London) 238, 225 (1972). 3. Wyatt, R. J., Murphy, D. L., Belmaker, R.. Cohen, S., Donnelly, C. H., and Pollin. W., Science 179, 916 (1972). 4. Meltzer, H., and Stuhl, S., Rrs. Commun. Chem. Pa&/. Pharmacol. 7, 419 (1974).
OXYGEN 5. 6. 7. 8.
AND HUMAN
PLATELET
MONOAMINE
OXIDASE
383
Collins, G. G. S., and Sandier, M., Biochem. Pkarmacol. 20, 289 (1971). Edwards, D. J., and Chang, S. S., Biochem. Biophys. Res. Commun. 65, 1018 (1975). Edwards. D. J., and Bums, M. O., Life Sci. 15, 1045 (1974). Eiduson, S., and Huang, R., in “Psychopharmacology Monograph” (E. Usdin, Ed.). Raven Press, New York (in press). 9. Philpot, F. J ., Biochem. J. 31, 856 (1937). IO. Kuhn, H. I., Biochem. J. 31, 1693 (1937). II. Novick, W. J., Biochem. Pharmacol. 15, 1009 (1966). 12. Roth, J. A., J. Neurochem. 27, 1107 (1976). 13. Huang, R., Eiduson, S., and Shih, J. C.. 1. Neurochem. 26, 799 (1976). 14. Lowry, 0. H., Rosebrough, N. J., Farr, A. L.. and Randall, R. 3. J. Biol. Chem. 193, 265 (1951). IS. White, H. L., and Wu, J. C., J. News-hem. 25, 21 (1976). 16. Huang, R., and Eiduson, S.. .I. Biol. Chem. 252, 284 (1977). 17. Friedman, E., Shopsin. B.. and Sathananthan, G., Amer. J. Psychid. 131, 1392 (1974).
MEDICINE
18, 378-383
(1977)
Effect of Oxygen on Human Platelet Monoamine Oxidase KIYOFUMI Neuropsychiatric Biobehavioral
KOBAYASHI'
AND SAMUEL EIDUSON
and Brain Research Institutes, Departments Sciences and Biological Chemistry, UCLA University of California at Los Angeles, Los Angeles, California 90024
of Psychiatry and Medical School,
Received April 5, 1977
The enzyme monoamine oxidase (MAO; E.C. 1.4.3.4.) is involved in the oxidative deamination of physiologically important monoamines (dopamine, norepinephrine, serotonin, and other biogenic amines) and is widely distributed in human tissues. Human blood platelets have been found to contain MAO (l), and a reduced MAO activity in platelets has recently been proposed as a genetic marker for vulnerability to schizophrenia (2-4). Although it has generally been accepted that MAO in other tissues such as brain and liver exists in multiple forms, this has not been thought to be the case for platelet MAO (5, 6). However, it has recently been reported that human platelet MAO contained multiple catalytic sites (6, 7). Furthermore, it has beeen observed in our laboratory, using a variety of techniques (8), that there may be at least two distinct conformations of human platelet MAO. It has also been reported that MAO activity in the liver, thyroid, heart, and brain of various animals was affected by the oxygen tension of the assay environment (9-l 1). More recently, Roth (12) has compellingly suggested that, at levels of oxygen of about 0.9 mM, phenethylamine and benzylamine “. . . share a common catalytic site . . .” and also bind to the reduced form of the enzyme. The present paper describes a study of the effect of oxygen on human platelet MAO using three different substrates at three different incubation temperatures and provides evidence that the catalytic site(s) of the human platelet enzyme may respond differentially to various substrates at different temperatures under these conditions. I Present address: Institute for Neurobiology, 2-5-l Shikata-cho, Okayama, Japan.
Okayama University Medical School,
378 Copyright All rights
@ 1977 by Academic Press, Inc. of reproduction in any form recerved
ISSN 0006-2944
OXYGEN
AND
HUMAN
PLATELET
MONOAMINE
OXIDASE
379
MATERIALS AND METHODS Human platelets were isolated from blood collected in citrate as described previously (13) and were stored in a freezer at -20°C. The isolated platelets were solubilized by sonication of the platelets for 5 set before assay. MAO activity was assayed radiochemically with three different substrates [side chain-2J4Cltryptamine bisuccinate and /3-El-L4Clphenylethylamine hydrochloride, New England Nuclear; [methylene J4C]benzylamine hydrochloride, ICN Pharmaceuticals, Inc.) and at three different temperatures (20, 37, and 42°C). Unlabeled substrates were obtained as follows: tryptamine hydrochloride and benzylamine from Sigma Chemical Co., /3-phenylethylamine from Eastman Kodak Co. The final concentrations of substrates were 1 x 10-5~. The enzyme preparation (in 0.5 M phosphate buffer, pH 7.4) was incubated at the appropriate temperature for 20 min. The reaction was then initiated by the addition of the substrate, and incubation was carried out by shaking for 5 min in air or by bubbling 100% oxygen into the solution for 5 set just after adding substrate. The oxygenated reaction tube was stoppered and shaken for 5 min. The reaction was stopped with the addition of 0.1 ml of 6 N HCl. The products were extracted with 6 ml of toluene. Protein determination was carried out by the method of Lowry et al. (14). Statistical significance was determined using the Mann-Whitney U test. RESULTS The effect of oxygen on human platelet MAO activity at three different temperatures when assayed with the substrates benzylamine, tryptamine, and phenylethylamine is summarized in Table 1. Using phenylethylamine as substrate, MAO activity significantly increased with oxygen at all three temperatures. MAO activity also increased with oxygen at 20 and 37°C when assayed with tryptamine as substrate. On the other hand, when benzylamine was used as substrate no alteration of enzyme activity with oxygen was observed at any temperature (see also Fig. 1). Figure 1 shows the ratio of MAO activity obtained in oxygen to that in air. The ratios of MAO activity when assayed with phenylethylamine were 1.90, 2.16, and 2.42 at 20, 37, and 42”C, respectively. (These values were significantly higher (P < 0.01) than those obtained with tryptamine and benzylamine.) The ratios of MAO activity with tryptamine as substrate were significantly higher (P < 0.01) than those for benzylamine at 20 and 37”C, but not at 42°C. Unlike that observed with phenylethylamine, increased temperature significantly decreased the oxygen effect when using tryptamine as substrate. The ratios with benzylamine, however, were not significantly different at three temperatures.
(12)
(12)
(12)
0.173 + 0.016 (17) 0.224 5 0.019
(12)
0.058 -e 0.003
Air
(12)
0.228 t 0.021* (17) 0.252 + 0.023
(12)
0.089 2 0.006**
Tryptamine
(12)
1.244 2 0.100 (17) 1.306 k 0.129
(12)
0.597 k 0.053
Air
1.142 + 0.123*” (12) 2.736 _f 0.272” ’ (17) 3.134 z 0.2x1-: (12)
Oxygen
_____
Phenylethylamine
TEMPERATURES”
of protein/5 min)
AT THREE
Oxygen
SUBSTRATES
“Data given are mean values t SEM, with the number of cases in parentheses. Significance of the difference of MAO activity in oxygen as compared to air: *P < 0.05, **P < 0.001 (Mann-Whitney U test).
42
0.245 2 0.023 (17) 0.322 2 0.029
(12)
(12)
0.245 + 0.023 (17) 0.329 k 0.029
0.093 + 0.007
0.087 k 0.009
20
37
1
THREE
TABLE USING
MAO activity @mole oxidizedimg
MAO ACTIVITY
Oxygen
ON
Air
Benzylamine
OF OXYGEN
Incubation temperature (“C)
EFFECT
OXYGEN
AND HUMAN
PLATELET
MONOAMINE
OXIDASE
381
%r-+-i+ temperature
(*C
1
FIG. I. The ratio of monoamine oxidase activity obtained in oxygen to that in air when assayed with the substrates benzylamine ( C---m), tryptamine (LO), and phenylethylamine (&A) at three temperatures. Each point represents the mean values k SEM, calculated from the ratio in individual cases (number of cases are indicated in Table 1).
DISCUSSION
It has been reported that increased oxygen tension of the assay environment increased the activity of MAO from various tissues such as thyroid, liver, heart, and brain (9-12). However, the rate of increase of MAO activity by oxygenation varies with tissues, species, and substrates used at 37°C (12). In the present study, we found that the platelet MAO activity increased with oxygenation of the reaction mixture when using phenylethylamine (at three temperatures) and tryptamine (20 and 37°C) as substrate, while there were no alterations in the activity when using benzylamine as substrate. The rates of increase were about 100% with phenylethylamine and 30% with tryptamine at 37°C. Furthermore, the increase of MAO activity varied with the temperature, both with phenylethylamine and tryptamine. Although human platelet MAO is electrophoretically homogenous (5, 6), it appears to have multiple binding or catalytic sites (6,7) as does brain mitochondrial MAO (15,16). Edwards and Chang (6) reported that human platelet MAO contained two interacting catalytic sites on the basis of
382
KOBAYASHJ
AND EJDUSON
substrate competition
experiments; i.e., one active site had a high affinity the other had a high affinity for benzylamine and tryptamine. On the other hand, Roth (13) suggested that both PEA and benzylamine occupy the same site on the B form of the enzyme derived from brain tissue. In our preliminary studies using both ESR and “Clabeled substrates, human platelet enzyme appears to exist in at least two forms or to have two different binding sites (8). Our present results are consistent with the existence of at least two catalytic or binding sites in the human platelet enzyme. While recent studies suggested that platelet MAO activity was reduced in schizophrenics and may be a genetic marker for vulnerability to schizophrenia (2-4), opposite results have also been reported by other workers (17). The reason for such contradictory results is not clear. However, if human platelet MAO has one or more binding sites as indicated above and which are affected by oxygen concentration, the possibility should be raised that it may not be appropriate to relate total MAO activity in platelets to behavior disorders by determining MAO activity using only one substrate in air and at one temperature (37°C). What may be more significant than total enzyme activity in regard to such disorders is the nature of the catalytic or binding sites of the enzyme. for phenylethylamine,
SUMMARY Effect of oxygen on the determination of human platelet monoamine oxidase (MAO) was studied by using three different substrates at three different temperatures. MAO activity using phenylethylamine as substrate increased about two times with oxygenation at all three temperatures. On the other hand, there was virtually no alteration in activity using benzylamine as substrate at any temperature. Using tryptamine as substrate, the rate of increase in MAO activity varied with the temperature used. Our findings suggest that the catalytic site(s) of the human platelet enzyme responds differentially to various substrates at different temperatures under different concentrations of oxygen. ACKNOWLEDGMENTS This work was supported by the Ralph E. Smith Foundation and National Institute of Mental Health Grant 19734-06. We are grateful to Ms. Ruth Chang for her skillful assistance.
REFERENCES 1. Paasonen, M. K., Solatunturi, E., and Kivalo, E., Psychopharmacologia 6, 120 (1964). 2. Murphy, D. L., and Wyatt, R., Nature (London) 238, 225 (1972). 3. Wyatt, R. J., Murphy, D. L., Belmaker, R.. Cohen, S., Donnelly, C. H., and Pollin. W., Science 179, 916 (1972). 4. Meltzer, H., and Stuhl, S., Rrs. Commun. Chem. Pa&/. Pharmacol. 7, 419 (1974).
OXYGEN 5. 6. 7. 8.
AND HUMAN
PLATELET
MONOAMINE
OXIDASE
383
Collins, G. G. S., and Sandier, M., Biochem. Pkarmacol. 20, 289 (1971). Edwards, D. J., and Chang, S. S., Biochem. Biophys. Res. Commun. 65, 1018 (1975). Edwards. D. J., and Bums, M. O., Life Sci. 15, 1045 (1974). Eiduson, S., and Huang, R., in “Psychopharmacology Monograph” (E. Usdin, Ed.). Raven Press, New York (in press). 9. Philpot, F. J ., Biochem. J. 31, 856 (1937). IO. Kuhn, H. I., Biochem. J. 31, 1693 (1937). II. Novick, W. J., Biochem. Pharmacol. 15, 1009 (1966). 12. Roth, J. A., J. Neurochem. 27, 1107 (1976). 13. Huang, R., Eiduson, S., and Shih, J. C.. 1. Neurochem. 26, 799 (1976). 14. Lowry, 0. H., Rosebrough, N. J., Farr, A. L.. and Randall, R. 3. J. Biol. Chem. 193, 265 (1951). IS. White, H. L., and Wu, J. C., J. News-hem. 25, 21 (1976). 16. Huang, R., and Eiduson, S.. .I. Biol. Chem. 252, 284 (1977). 17. Friedman, E., Shopsin. B.. and Sathananthan, G., Amer. J. Psychid. 131, 1392 (1974).
