Acta psychiat. scand. (1976) 54, 67-72 The Jerusalem Mental Health Center - Ezrath Nashim (Head: Prof. M . Rosenbnurn), Jerusalem, Israel
Electrophoresis of platelet monoarnine oxidase in schizophrenia and manic-depressiveillness R. H. BELMAKER,R. EBSTEIN,R. RIMON,R. J. WYATT AND D. L. MURPHY Monoamine oxidase is an important enzyme in the catabolism of biogenic amines and can be measured in human platelets. Platelet M A 0 has been reported to be reduced in schizophrenic and manicdepressive patients, though other reports are contradictory. The present study evaluated the possibility that qualitative genetic enzyme abnormalities of MA0 could be responsible for the different enzyme activities of platelet MA0 in different populations. However, polyacrylamide gel electrophoresis of platelet MA0 from 10 manic-depressive, 12 schizophrenic, and 11 normal individuals did not reveal any genetic mutant forms.
Key words: Monoamine oxidase - schizophrenia - mania - depression - platelet - electrophoresis. Monoamine oxidase (MAO) is an important enzyme in the catabolism of brain biogenic amines (Kopin (1964)). Human platelets contain an intramitochondrial M A 0 that is similar in many physiochemical properties to brain M A 0 (Murphy et al. (1974)). There are large differences between human individuals in the levels of activity of the platelet M A 0 enzyme, and these individual differences seem to be largely genetic in origin (Nies et al. (1973)). Schizophrenic (Murphy & Wyatt (1972)) and manic-depressive patients (Murphy & Weiss (1972)) have been reported as having low platelet MAO. Reduced platelet M A 0 activity has been felt to be a possible genetic marker in schizophrenia because of the results of a study of monozygotic twins discordant for schizophrenia (Wyatt et al. (1973)). In this study of monozygotic discordant twins the schizophrenic twin and his healthy co-twin both had reduced platelet M A 0 activity, indicating that this finding was not due to hospitalization, psychoactive drugs, or other secondary effects of illness. Attempts to replicate the original findings of reduced platelet MA0 in schizophrenia and manic-depressive illness have led to conflicting results. Meltzer & Stahf (1974) confirmed the finding of low platelet M A 0 in schizophrenia, but Brockington et al. (1976) could not. Carpenter et al. (1975) did not find reduced platelet M A 0 activity in acute schizophrenic patients, and Friedman et al. (1974) found normal platelet M A 0 activity in newly admitted patients with recurrent schizophrenia. Belmaker et al. (1976) in Jerusalem found normal platelet MA0 activity in chronic schizophrenic patients but significantly increased platelet
68 M A 0 activity in bipolar manic-depressive patients. Leckman et al. (1976) replicated in the USA the original finding of reduced platelet M A 0 activity in bipolar manic-depressive patients. One possible explanation of these conflicting results would be the existence of population differences in an underlying, qualitative enzyme abnormality. Sickle disease hemoglobin is a major cause of anemia in East Africa, for instance, but hemoglobin C disease is a common cause of anemia in West Africa (Harris (1975)). Another example exists in the numerous mutant forms of glucose-6phosphate-dehydrogenase (G6PD), some of which have reduced enzyme activity when measured in vitro whereas others have normal or even increased enzyme activity (Yoshida (1973)). Some of the mutant forms are associated with hemolytic anemia. The technique of polyacrylamide gel electrophoresis (PAGE) is capable of revealing qualitative differences between proteins arising from genetic variation at the molecular level. Such variant enzymes are often detectable by differences in their rate of migration during electrophoresis. Harris (1975) has estimated that over 25 % of enzyme proteins will show genetic mutant forms or polymorphism in a manner detectable by electrophoresis. We therefore decided to examine using PAGE platelet MA0 from manic-depressive and schizophrenic patients. MATERIAL AND METHODS Patients were selected from the inpatient and outpatient facilities of the Jerusalem Mental Health Center. Diagnoses were made according to the criteria of Feighner et al. (1972)). Patients were diagnosed as manic-depressive if they had had at least one episode of mania. Schizophrenic patients were chronic patients with hallucinations or delusions, as well as inappropriate affect, loose associations, and at least 2 years’ hospitalization. Since psychoactive drugs had been reported not to affect platelet M A 0 activity (Murphy et al. (1974)), patients were sampled without regard to medication. All schizophrenic patients were receiving phenothiazine medication at the time of sampling, and all manic-depressive patients were receiving lithium. No patient was receiving an MA0 inhibitor. Controls were volunteer hospital staff members with no history of mental illness. The manic-depressive patients comprised four males and six females with a mean age of 44, the schizophrenic patients comprised seven males and five females with a mean age of 28, and the normal controls comprised seven males and four females with a mean age of 30. Blood was drawn by venipuncture and platelet pellets were prepared as described previously (Murphy et al. (1974), Belmaker et al. (1976)). The platelet pellet was then sonicated in 1.0 cm3 of 1 % Triton X-100 for 5 sec and stored at -7OOC for not more than 2 weeks before electrophoresis. The length of sonication of up to 1 hour was not found to alter the pattern of platelet M A 0 on PAGE. Collins & Sandler (1971) and Edwards & Chang (1975) have previously reported on the PAGE of platelet MA0 from sonicated platelet preparations. The present method used a 5 % polyacrylamide gel without stacking segment (Davis (1964), Ornstein (1964)). The gels were run in 0.4 M glycine tris buffer,
69
persulfate, a 50-pl sample of platelet sonicate mixed with 20 pl of 50 % sucrose was applied to the top of the PAGE column. Electrophoresis was carried out for approximately 90 min, the exact time being determined by the running time of a bromphenol blue dye marker. Gels were cut by hand after electrophoresis into 2-mm sections and each section assayed the same day for M A 0 activity. The assay used 14C-benzylamine as substrate (Nuclear Research Chemicals, 4 mCimmo1) because this substrate has the highest V ,, for platelet M A 0 (Robinson et al. (1963)). To each gel slice was added 0.15 pCi of 14C-benzylamine in 0.5 ml of 0.2 M tris buffer, pH 9.1, as described by Belmaker et al. (1976). The W!-benzylamine was not, however, diluted with nonradioactive benzylamine, thus permitting maximum production of labeled product by the enzyme. The modified assay, while operating below the optimal K, for MAO, allowed the detection of the amounts of M A 0 activity present after PAGE of unpurified platelet sonicates from individual patients. The remainder of the assay was carried out as described by Belmaker et al. (1976), except that the incubation time was 60 min instead of 30 min. M inhibited 100 % of the Preincubation of gel slices with pargyline at enzyme activity on the gel. Approximately 35 % of the platelet M A 0 activity applied on the gel was recoverable after electrophoresis in the various gel slices assayed.
a 0
a I
Fig. 1. Mean electrophoretic pattern of platelet M A 0 from schizophrenic, manic-depressive, and control individuals.
70 RESULTS Fig. 1 shows the mean PAGE pattern for each group for 10 manic-depressive, pH 8.3, at 5OC using 3 mA/gel. After a 45-min prerun to remove ammonium 12 schizophrenic, and 11 normal individuals. After PAGE two peaks of platelet M A 0 activity are detectable, one migrating 8-10 mm and the other broader peak migrating 24-32 mm into the gel in the time allotted. There are, however, no differences in the rate of migration of either band between the groups examined, and no individual was found with a peak in a reproducibly altered position on the gel at the pH used (pH 8.3). The increased platelet M A 0 activity found in manic-depressive patients in Jerusalem (Belmaker et al. (1976)) is clearly apparent even after PAGE in both electrophoretic peaks. However, this increased height of each peak of the manic-depressives does not give any information about mutant forms, since no variation in the rate of migration of the peaks was observed. No individual patient was observed to have an absence of either electrophoretic peak of platelet MAO.
DISCUSSION The present study did not identify any mutant or polymorphic forms of human platelet MAO. Unlike other proteins such as hemoglobin or G6PD, platelet M A 0 would seem to be qualitatively similar in all 33 individuals examined and in all three psychiatric diagnostic groups. The absence of rate variants of the enzyme in PAGE suggests an absence of genetic DNA variants in the gene that codes for platelet MAO. Genetic variation in the activity of the platelet M A 0 enzyme between human individuals (Nies et al. (1973)) might therefore be based on other factors, such as differences in the rate of inactivation of the enzyme. The finding of two electrophoretic bands of platelet M A 0 confirms the recent study of Edwards & Chang (1975). A previous study (Collins & Sandler (1971)) that found platelet M A 0 to be electrophoretically homogeneous used purified enzyme that may have undergone changes in physiochemical properties. Edwards & Chang (1975) suggest that the slower peak of platelet M A 0 may represent an aggregate, though we did not find differences in the two peaks with varying amounts of sonication. The present lack of success in finding rate variants of platelet M A 0 in PAGE should not be accepted as conclusive. The technical problems are formidable, as platelet M A 0 is a relatively large molecule (mol.wt. 235,000) and is also membrane-bound (Collins & Sandler (1971)). The quantity of M A 0 enzyme activity available by peripheral venipuncture in individual psychiatric patients is too small to allow purification before PAGE. In the present study the platelet M A 0 was solubilized for PAGE as gently as possible, with 5 sec of sonication in 1 % Triton X-100 as detergent, but no Triton was used in the gel or buffer solution itself. We were afraid that an excess of Triton would coat all proteins present and prevent the appearance of differences in migration rate due to small
71 charge differences. However, it is possible that even the minimal amount of Triton used obscured charge differences and resulted in electrophoretic mobility dependent solely on size. New methods need to be developed to “clean” MA0 of its lipid attachments without coating the enzyme with Triton-like detergents. Electrophoresis of such a clean enzyme might yield more information about the characteristics of the enzyme, including possible mutant forms. Another important consideration that moderates the negative impact of this study is the influence of pH on PAGE (Harris (1975)). Some mutant enzyme forms become apparent in PAGE at specific pH levels only, and further electrophoretic screening of human platelet M A 0 at pH levels other than pH 8.3 is therefore necessary. Further study is also necessary to define why one band of platelet MA0 activity in this study was considerably broader on PAGE than the other band. This might imply heterogeneity of lipid attachments in the broad band of MAO, though the length of sonication did not affect the sharpness of the broad band. The relationship of platelet and brain M A 0 has been a subject of considerable controversy. Multiple electrophoretic forms of M A 0 have been reported in brain (Youdim (1973)), which suggests that platelet M A 0 characteristics could scarcely have general relevance for brain MAO. Tipton et al. (1973),however, have reported that the multiple electrophoretic forms of brain M A 0 can be reduced to one form by chemical treatment. This implies that perhaps only one M A 0 gene product exists, a M A 0 protein. Other researchers have divided M A 0 into Type A and Type B, according to inhibition sensitivity with clorgyline or deprenyl (Nefl & Goridis (1972)). Platelet M A 0 is a Type B enzyme in this classification whereas “brain-specific” M A 0 is Type A (Murphy & Donnelly (1974)). Inhibitor sensitivity, however, may also be affected by lipid attachments, as perchlorate treatment abolishes differences in response to clorgyline (Tipton et al. (1973)). In some enzyme systems, such as that of Tay-Sachs disease or homocystinuria, the peripheral enzyme is indeed the same gene product as the enzyme in brain, and medical research and diagnosis have benefited from this discovery (Uhlendorf & Mudd (1968), Okada & O’Brien (1969)). A summary of the present state of platelet M A 0 in schizophrenia and manicdepressive illness should convey both the modesty of the findings and the excitement of the field. Platelet M A 0 activity may be reduced in schizophrenia, and elevated or reduced in manic-depressive illness, though others find no such changes. We have not identified any mutant forms of human platelet MAO. On the other hand, schizophrenia and manic-depressive illness are partially genetic illnesses (Rosenthal (1970)) and modern genetics is largely biochemical genetics. Peripheral M A 0 may provide a window to a gene that controls a major enzyme in central catecholamine metabolism.
REFERENCES Belmaker, R . H., K . Elbesen, R . Ebstein & R . Rimon (1976):Platelet monoamine oxi-
dase in Schizophrenia and manic-depressive illness. Brit. J. Psychiat., in press. Brockington, I., T . J . Crow, E. C . Johnstone & F. Owen (1976): An investigation of platelet monoamine oxidase in schizophrenia and schizeaffective psychosis. In
press.
72 Carpenter, W . T.,Jr., D. L . Murphy & R . I . Wyatt (1975): Platelet monoamine oxidase activity in acute schizophrenia. Amer. J. Psychiat. 132, 438441. Collins, G . G. S., & M . Sandler (1971): Human blood platelet monoamine oxidase. Biochem. Pharmacol. 20, 289-2%. Davis, B. (1964): Disc electrophoresis, Part 2. Ann. N. Y. Acad. Sci. 121, 404-427. Edwards, D . J., & S. S. Chang (1975): Evidence for interacting catalytic sites of human platelet monoamine oxidase. Biochem. biophys. Res. Comm. 65, 1018-1025. Feighner, J . P., E. Robins, S. B. Guze, R . A . Woodrufl,Jr., G . Winokur & R . Munoz (1972): Diagnostic criteria for use in psychiatric research. Arch. gen. Psychiat. 26, 57-63. Friedman, E., B. Shopsin, G . Sathananthan & S. Gershon (1974): Blood platelet monoamine oxidase activity in psychiatric patients. Amer. J. Psychiat. 131, 1392-1394. Harris, H . (1975): The principles of human biochemical genetics. 2nd Ed. North-Holland Publishing Co., Amsterdam. Kopin, I . J. (1964): Storage and metabolism of catecholamines: The role of monoamine oxidase. Pharmacol. Rev. 16, 179-191. Leckman, J . F., E. S. Gershon, D . L. Murphy & A . S . Nichols (1976): Reduced platelet monoamine oxidase activity in first degree relatives of individuals with bipolar affective disorders. In press. Meltzer, H . Y., & S. M . Stahi (1974): Platelet monoamine oxidase activity and substrate preferences in schizophrenic patients. Res. Commun. Path. Pharmacol. 7,419-431. Murphy, D . L., R . H . Belmaker & R . 1. Wyatt (1974): Monoamine oxidase in schizophrenia and other behavioral disorders. J. psychiat. Res.11, 221-247. Murphy, D . L., & C. H . Donnelly (1974): Monoamine oxidase in man. In Usdin, E. (ed.): Neuropsychopharmacology of monoamines and their regulatory enzymes. Vol. 12. Advances in Biochemical Pharmacology. Raven Press, New York, pp. 71-85. Murphy, D . L., & R . Weiss (1972): Reduced monoamine oxidase activity in blood platelets from bipolar depressed patients. h e r . J. Psychiat. 128, 1351-1357. Murphy, D . L., & R . J . Wyatt (1972): Reduced MA0 activity in blood platelets from schizophrenic patients. Nature (Lond.) 238, 225-226. N e f f , N .H., & C . Goridis (1972): Neuronal monoamine oxidase. In Costa, E., & P . Greengard (ad.): Advances in Biochemical Pharmacology. Vol. 5 . Raven Press, New York, pp. 307-323. Nies, A., D . S. Robinson, K . R. Lamborn & R . P . Lampert (1973): Genetic control of platelet and plasma monoamine oxidase activity. Arch. gen. Psychiat. 28, 834-838. Okada, S., & J . S. O’Brien (1969): Tay-Sachs disease: Generalized absence of a B-D-Nacetylhexosaminidase component. Science 165, 698-701. Omstein, L. (1964): Disc electrophoresis, Part 1. Ann. N.Y. Acad. Sci. 121, 321-335. Robinson, D . S., W . Lovenberg, H . Keiser & J . Sjoerdsma (1968): Effects of drugs on human blood platelet and plasma amine oxidase in vitro and in vivo. Biochem. Pharmacol. 17, 109-119. Rosenthal, D . (1970): Genetic theory and abnormal behavior. McGraw-Hill, New York. Tipton, K . F., M . D . Houslay & N . I . Garrett (1973): Allotopic properties of human brain monoamine oxidase. Nature New Biol. 246, 213-214. Uhlendorf, W . B., & H . S. Mudd (1968): Cystathionine synthase in tissue culture derived from human skin: enzyme defect in homocystinuria. Science 160, 1007-1009. Wyatt, R . J., D . L. Murphy, R . H . Belmaker, S. Cohen, C . H . Donnelly & W . Pollin (1973): Reduced monoamine oxidase in platelets: A possible genetic marker for vulnerability to schizophrenia. Science 179, 916-917. Yoshida, A . (1973): Hemolytic anemia and G6PD deficiency. Science 179, 532-537. Youdim, M . B. H . (1973): Multiple forms of mitochondrial monoamine oxidase. Brit. med. Bull. 29, 120-122.
73 Received March 25, 1976
Robert H . Belmaker, M.D." Richard Ebstein, Ph.D. Ranan Rimon, M.D. The Jerusalem Mental Health Center Ezrath Nashim P.O.B. 140 Jerusalem Israel R . J . Wyatt D. L. Murphy N.I.M.H., Bethe-sda, Md., USA
*
Address for reprint requests.
Electrophoresis of platelet monoarnine oxidase in schizophrenia and manic-depressiveillness R. H. BELMAKER,R. EBSTEIN,R. RIMON,R. J. WYATT AND D. L. MURPHY Monoamine oxidase is an important enzyme in the catabolism of biogenic amines and can be measured in human platelets. Platelet M A 0 has been reported to be reduced in schizophrenic and manicdepressive patients, though other reports are contradictory. The present study evaluated the possibility that qualitative genetic enzyme abnormalities of MA0 could be responsible for the different enzyme activities of platelet MA0 in different populations. However, polyacrylamide gel electrophoresis of platelet MA0 from 10 manic-depressive, 12 schizophrenic, and 11 normal individuals did not reveal any genetic mutant forms.
Key words: Monoamine oxidase - schizophrenia - mania - depression - platelet - electrophoresis. Monoamine oxidase (MAO) is an important enzyme in the catabolism of brain biogenic amines (Kopin (1964)). Human platelets contain an intramitochondrial M A 0 that is similar in many physiochemical properties to brain M A 0 (Murphy et al. (1974)). There are large differences between human individuals in the levels of activity of the platelet M A 0 enzyme, and these individual differences seem to be largely genetic in origin (Nies et al. (1973)). Schizophrenic (Murphy & Wyatt (1972)) and manic-depressive patients (Murphy & Weiss (1972)) have been reported as having low platelet MAO. Reduced platelet M A 0 activity has been felt to be a possible genetic marker in schizophrenia because of the results of a study of monozygotic twins discordant for schizophrenia (Wyatt et al. (1973)). In this study of monozygotic discordant twins the schizophrenic twin and his healthy co-twin both had reduced platelet M A 0 activity, indicating that this finding was not due to hospitalization, psychoactive drugs, or other secondary effects of illness. Attempts to replicate the original findings of reduced platelet MA0 in schizophrenia and manic-depressive illness have led to conflicting results. Meltzer & Stahf (1974) confirmed the finding of low platelet M A 0 in schizophrenia, but Brockington et al. (1976) could not. Carpenter et al. (1975) did not find reduced platelet M A 0 activity in acute schizophrenic patients, and Friedman et al. (1974) found normal platelet M A 0 activity in newly admitted patients with recurrent schizophrenia. Belmaker et al. (1976) in Jerusalem found normal platelet MA0 activity in chronic schizophrenic patients but significantly increased platelet
68 M A 0 activity in bipolar manic-depressive patients. Leckman et al. (1976) replicated in the USA the original finding of reduced platelet M A 0 activity in bipolar manic-depressive patients. One possible explanation of these conflicting results would be the existence of population differences in an underlying, qualitative enzyme abnormality. Sickle disease hemoglobin is a major cause of anemia in East Africa, for instance, but hemoglobin C disease is a common cause of anemia in West Africa (Harris (1975)). Another example exists in the numerous mutant forms of glucose-6phosphate-dehydrogenase (G6PD), some of which have reduced enzyme activity when measured in vitro whereas others have normal or even increased enzyme activity (Yoshida (1973)). Some of the mutant forms are associated with hemolytic anemia. The technique of polyacrylamide gel electrophoresis (PAGE) is capable of revealing qualitative differences between proteins arising from genetic variation at the molecular level. Such variant enzymes are often detectable by differences in their rate of migration during electrophoresis. Harris (1975) has estimated that over 25 % of enzyme proteins will show genetic mutant forms or polymorphism in a manner detectable by electrophoresis. We therefore decided to examine using PAGE platelet MA0 from manic-depressive and schizophrenic patients. MATERIAL AND METHODS Patients were selected from the inpatient and outpatient facilities of the Jerusalem Mental Health Center. Diagnoses were made according to the criteria of Feighner et al. (1972)). Patients were diagnosed as manic-depressive if they had had at least one episode of mania. Schizophrenic patients were chronic patients with hallucinations or delusions, as well as inappropriate affect, loose associations, and at least 2 years’ hospitalization. Since psychoactive drugs had been reported not to affect platelet M A 0 activity (Murphy et al. (1974)), patients were sampled without regard to medication. All schizophrenic patients were receiving phenothiazine medication at the time of sampling, and all manic-depressive patients were receiving lithium. No patient was receiving an MA0 inhibitor. Controls were volunteer hospital staff members with no history of mental illness. The manic-depressive patients comprised four males and six females with a mean age of 44, the schizophrenic patients comprised seven males and five females with a mean age of 28, and the normal controls comprised seven males and four females with a mean age of 30. Blood was drawn by venipuncture and platelet pellets were prepared as described previously (Murphy et al. (1974), Belmaker et al. (1976)). The platelet pellet was then sonicated in 1.0 cm3 of 1 % Triton X-100 for 5 sec and stored at -7OOC for not more than 2 weeks before electrophoresis. The length of sonication of up to 1 hour was not found to alter the pattern of platelet M A 0 on PAGE. Collins & Sandler (1971) and Edwards & Chang (1975) have previously reported on the PAGE of platelet MA0 from sonicated platelet preparations. The present method used a 5 % polyacrylamide gel without stacking segment (Davis (1964), Ornstein (1964)). The gels were run in 0.4 M glycine tris buffer,
69
persulfate, a 50-pl sample of platelet sonicate mixed with 20 pl of 50 % sucrose was applied to the top of the PAGE column. Electrophoresis was carried out for approximately 90 min, the exact time being determined by the running time of a bromphenol blue dye marker. Gels were cut by hand after electrophoresis into 2-mm sections and each section assayed the same day for M A 0 activity. The assay used 14C-benzylamine as substrate (Nuclear Research Chemicals, 4 mCimmo1) because this substrate has the highest V ,, for platelet M A 0 (Robinson et al. (1963)). To each gel slice was added 0.15 pCi of 14C-benzylamine in 0.5 ml of 0.2 M tris buffer, pH 9.1, as described by Belmaker et al. (1976). The W!-benzylamine was not, however, diluted with nonradioactive benzylamine, thus permitting maximum production of labeled product by the enzyme. The modified assay, while operating below the optimal K, for MAO, allowed the detection of the amounts of M A 0 activity present after PAGE of unpurified platelet sonicates from individual patients. The remainder of the assay was carried out as described by Belmaker et al. (1976), except that the incubation time was 60 min instead of 30 min. M inhibited 100 % of the Preincubation of gel slices with pargyline at enzyme activity on the gel. Approximately 35 % of the platelet M A 0 activity applied on the gel was recoverable after electrophoresis in the various gel slices assayed.
a 0
a I
Fig. 1. Mean electrophoretic pattern of platelet M A 0 from schizophrenic, manic-depressive, and control individuals.
70 RESULTS Fig. 1 shows the mean PAGE pattern for each group for 10 manic-depressive, pH 8.3, at 5OC using 3 mA/gel. After a 45-min prerun to remove ammonium 12 schizophrenic, and 11 normal individuals. After PAGE two peaks of platelet M A 0 activity are detectable, one migrating 8-10 mm and the other broader peak migrating 24-32 mm into the gel in the time allotted. There are, however, no differences in the rate of migration of either band between the groups examined, and no individual was found with a peak in a reproducibly altered position on the gel at the pH used (pH 8.3). The increased platelet M A 0 activity found in manic-depressive patients in Jerusalem (Belmaker et al. (1976)) is clearly apparent even after PAGE in both electrophoretic peaks. However, this increased height of each peak of the manic-depressives does not give any information about mutant forms, since no variation in the rate of migration of the peaks was observed. No individual patient was observed to have an absence of either electrophoretic peak of platelet MAO.
DISCUSSION The present study did not identify any mutant or polymorphic forms of human platelet MAO. Unlike other proteins such as hemoglobin or G6PD, platelet M A 0 would seem to be qualitatively similar in all 33 individuals examined and in all three psychiatric diagnostic groups. The absence of rate variants of the enzyme in PAGE suggests an absence of genetic DNA variants in the gene that codes for platelet MAO. Genetic variation in the activity of the platelet M A 0 enzyme between human individuals (Nies et al. (1973)) might therefore be based on other factors, such as differences in the rate of inactivation of the enzyme. The finding of two electrophoretic bands of platelet M A 0 confirms the recent study of Edwards & Chang (1975). A previous study (Collins & Sandler (1971)) that found platelet M A 0 to be electrophoretically homogeneous used purified enzyme that may have undergone changes in physiochemical properties. Edwards & Chang (1975) suggest that the slower peak of platelet M A 0 may represent an aggregate, though we did not find differences in the two peaks with varying amounts of sonication. The present lack of success in finding rate variants of platelet M A 0 in PAGE should not be accepted as conclusive. The technical problems are formidable, as platelet M A 0 is a relatively large molecule (mol.wt. 235,000) and is also membrane-bound (Collins & Sandler (1971)). The quantity of M A 0 enzyme activity available by peripheral venipuncture in individual psychiatric patients is too small to allow purification before PAGE. In the present study the platelet M A 0 was solubilized for PAGE as gently as possible, with 5 sec of sonication in 1 % Triton X-100 as detergent, but no Triton was used in the gel or buffer solution itself. We were afraid that an excess of Triton would coat all proteins present and prevent the appearance of differences in migration rate due to small
71 charge differences. However, it is possible that even the minimal amount of Triton used obscured charge differences and resulted in electrophoretic mobility dependent solely on size. New methods need to be developed to “clean” MA0 of its lipid attachments without coating the enzyme with Triton-like detergents. Electrophoresis of such a clean enzyme might yield more information about the characteristics of the enzyme, including possible mutant forms. Another important consideration that moderates the negative impact of this study is the influence of pH on PAGE (Harris (1975)). Some mutant enzyme forms become apparent in PAGE at specific pH levels only, and further electrophoretic screening of human platelet M A 0 at pH levels other than pH 8.3 is therefore necessary. Further study is also necessary to define why one band of platelet MA0 activity in this study was considerably broader on PAGE than the other band. This might imply heterogeneity of lipid attachments in the broad band of MAO, though the length of sonication did not affect the sharpness of the broad band. The relationship of platelet and brain M A 0 has been a subject of considerable controversy. Multiple electrophoretic forms of M A 0 have been reported in brain (Youdim (1973)), which suggests that platelet M A 0 characteristics could scarcely have general relevance for brain MAO. Tipton et al. (1973),however, have reported that the multiple electrophoretic forms of brain M A 0 can be reduced to one form by chemical treatment. This implies that perhaps only one M A 0 gene product exists, a M A 0 protein. Other researchers have divided M A 0 into Type A and Type B, according to inhibition sensitivity with clorgyline or deprenyl (Nefl & Goridis (1972)). Platelet M A 0 is a Type B enzyme in this classification whereas “brain-specific” M A 0 is Type A (Murphy & Donnelly (1974)). Inhibitor sensitivity, however, may also be affected by lipid attachments, as perchlorate treatment abolishes differences in response to clorgyline (Tipton et al. (1973)). In some enzyme systems, such as that of Tay-Sachs disease or homocystinuria, the peripheral enzyme is indeed the same gene product as the enzyme in brain, and medical research and diagnosis have benefited from this discovery (Uhlendorf & Mudd (1968), Okada & O’Brien (1969)). A summary of the present state of platelet M A 0 in schizophrenia and manicdepressive illness should convey both the modesty of the findings and the excitement of the field. Platelet M A 0 activity may be reduced in schizophrenia, and elevated or reduced in manic-depressive illness, though others find no such changes. We have not identified any mutant forms of human platelet MAO. On the other hand, schizophrenia and manic-depressive illness are partially genetic illnesses (Rosenthal (1970)) and modern genetics is largely biochemical genetics. Peripheral M A 0 may provide a window to a gene that controls a major enzyme in central catecholamine metabolism.
REFERENCES Belmaker, R . H., K . Elbesen, R . Ebstein & R . Rimon (1976):Platelet monoamine oxi-
dase in Schizophrenia and manic-depressive illness. Brit. J. Psychiat., in press. Brockington, I., T . J . Crow, E. C . Johnstone & F. Owen (1976): An investigation of platelet monoamine oxidase in schizophrenia and schizeaffective psychosis. In
press.
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73 Received March 25, 1976
Robert H . Belmaker, M.D." Richard Ebstein, Ph.D. Ranan Rimon, M.D. The Jerusalem Mental Health Center Ezrath Nashim P.O.B. 140 Jerusalem Israel R . J . Wyatt D. L. Murphy N.I.M.H., Bethe-sda, Md., USA
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