Biochemical Pharmacology, Vol. 28, pp. 3063-3068 0 Pergamon Press Ltd. 1979. Printed in Great Britain. 0006.2952/79/1015-3063 $02.00/O zyxwv THE INTERACTION BETWEEN HUMAN PLATELET MONOAMINE OXIDASE, ITS MONOAMINE SUBSTRATES AND OXYGEN CHRISTOPHER ~.FOWLER,* BERTIL EKSTEDT,TORU EGASHIRA,-~ HIROYASU K~EMU~HI~ and LARS ORELAND Department of Pharmacology, University of Umea S-901 87 Urn&, Sweden zyxwvutsrqponmlkjihgfe (Received 23 February 1979; accepted 30 April 1979) Abstract-The interaction of human platelet MAO-B with three substrates @phenethylamine, tryptamine and benzylamine) has been investigated in an attempt to determine whether or not there exists heterogeneity of this enzyme form. Treatment with pargyline, thermal denaturati~ and 2-butanone affected the enzyme activity to the same degree, regardless of the amine substrate used. Mixed substrate experiments indicated that the substrates inhibited each other in a competitive manner with Ki values close to their K, values. The activity of MAO-B was increased in an uncompetitive manner as the oxygen concentration was raised. However, the degree of this increase was dependent upon the amine substrate used to assay for activity. These results are consistent for an enzvme with a single binding site for amine substrates and a possible multiplicity of binding sites for oxygen. In the study of psychiatric disorders, great attention has been drawn to the turnover of monoamine transmitter agents and to the activities of the enzymes involved in their metabolism. Monoamine oxidase (MAO) (EC 1.4.3.4) is an enzyme of major importance for the catabolism of, e.g. noradrenaline, dopamine and S- hydroxy~yp~mine [ 1, 21. The turnover of Shydroxy- tryptamine in brain as estimated by the levels of 5- hydroxytryptamine and 5-hydroxyindoleacetic acid in autopsy material has recently been found to correlate with the activity of MAO in brain [ 3 1. Furthermore, there are indications that platelet MAO activity is also correlated with serotonin turnover in the brain since a correlation has recently been found between platelet MAO activity and levels of Shydroxyindole acetic acid in the cerebrospinal fluid of healthy volunteers 141. As the human blood platelet is a convenient source of human MAO, a great number of studies on the activity of this enzyme have been performed in a variety of psychiatric disorders [for reviews see 5, 61. On the basis of the differential sensitivity to inhibi- tion of MAO by the acetylenic inhibitor clorgyline when assayed with different substrates, the activity of MAO has been divided into two forms, MAO-A and B, the A form being inhibited by much lower concentra- tions of clorgyline [ 7, for review see 81. In human blood platelets. only the B form of MAO is present [91. However, on the basis of mixed substrate experiments and studies on the mechanisms of inhibition by tricyclic antidepressants, it has been suggested that there is more than one catalytic site for the deamination of mono- amines by human blood platelet MAO-B, with one preferentially oxidizing benzylamine and tryptamine and the other ~-phene~yl~ine [ 10, 111. In this study, an attempt has been made to explore - * To whom correspondence should be addressed. t Present address: Showa University, School of Medicine, Department of Pharmacology, Hatanodai I-5-8, Shinagawa- ku, Tokyo, Japan. the nature of human blood platelet MAO in order to determine whether or not such multiplicity of catalytic sites exists. MATERIALS AND METHODS P~e~~~~o~ of blood ~l~~e~e~s. Blood samples (450ml in a standard buffered citrate solution) were obtained from male volunteers (aged between 24 and 42). The blood was centrifuged at 200 g for 10 min to remove the red cells. The cells were washed twice with 0.15 M NaCl and ,the combined supernatants further centrifuged at 13,000 g for 15 min to produce a platelet preparation. The pellet was resuspended in 5.0 ml of 0.01 M potassium phosphate (pH 7.4) and stored at -80”. Before use, the suspension was sonicated in an MSE Ultrasonic Disintegrator at low power for 2 min, to produce a more homogeneous preparation. The properties of rat liver MAO have been shown not to be changed when sonicated in this manner i 121. Lipid depletion of blood platelets was carried out as described previously I 131.Over a period of 5 min, 8 ml of 2-butanone was added to 1 ml of a blood platelet preparation, the suspension being kept at +4’ and stirred continuously. This suspension was centrifuged at 20,000 g for 5 min, the pellet resuspended in 0.0 1 M potassium phosphate buffer (PH 7.4), stirred for 5 min at +4’, then recentrifuged and resuspended as before. Aliquots of the pellet and supernatant fractions were assayed for MAO activity. Monoamine oxidase assay. Monoamine oxidase ac- tivity was assayed radiochemically by two different methods, both of which gave essentially similar results. For the mixed substrate and ‘oxygen ratio’ex~riments, the assay described by C~iin~~ and Laverty I141 was used, with two modifications. The buffer solution used in the assay mixture in these experiments was 0.25 M sucrose, buffered with 0.0 1 M potassium phos- phate (pH 7.8), a lower ionic strength buffer than used previously, as the ionic strength of buffer solutions has been shown to influence the activity of MAO [ 153. 3063