Abstract
In this study, we investigated the microdetermination of human platelet monoamine oxidase (MAO) activity and compared the previous method for determination of platelet MAO activity. As the enzyme materials, platelet rich plasma (PRP) that was collected without centrifugation and with centrifugation at 175×g (centrifuged PRP) and platelet pellet that was collected with recentrifugation of the centrifuged PRP at 2000×g were used. Platelet MAO activity was assayed radiometrically. The effects of clorgyline, specific inhibitor of type A MAO, and deprenyl, specific inhibitor of type B MAO, on MAO in semicarbazide-pretreated PRP were examined using tyramine, benzylamine and β-phenylethylamine (PEA) as substrates. MAO activities were inhibited by high concentrations of clorgyline, but inhibited by low concentrations of deprenyl and inhibition curves were single sigmoidal with these three substrates. From these results, it is confirmed that almost only type B MAO is contained in human platelet. Specific activities of platelet MAO were 13.3, 11.6, 3.11 and 0 n mole/hr/mg protein using tyramine, benzylamine, PEA and 5-hydroxytryptamine (5-HT) as substrates. The effects of semicarbazide, specific inhibitor of plasma amine oxidase, and pargyline, specific inhibitor of platelet MAO on MAO activity in PRP were examined since plasma and platelet are included in PRP. Semicarbazide did not inhibit MAO activity at all, while pargyline completely inhibited its activity at concentration of 1μM using tyramine as substrate. Using benzylamine as substrate, platelet MAO activity remained about 10% of control by pargyline at a concentration of 1μM. However, MAO activity was completely inhibited by pargyline at a concentration of 10μM when the enzyme preparation was pretreated with 1mM semicarbazide. These results indicate that plasma amine oxidase activity is negligible when enzyme material is pretreated with semicarbazide. MAO activity in PRP, centrifuged PRP and platelet pellet were examined using tyramine as substrate. The dpm/number of platelets were 2.04×10-3, 2.07×10-3and 0.65×10-3and the dpm values were 2437, 1074 and 1537 using PRP, centrifuged PRP and platelet pellet, respectively. The relationship between platelet MAO activity and number of platelets was examined in 54 normal subjects. The number of platelets were from 45 to 78×104/μl and platelet MAO activity ranged from 523 to 2593 dpm. Reciprocity was y=11.2x+690.4 and the correlation coefficient was 0.784. These results indicate that our microdetermination method of human platelet MAO activity is easy to assay and it requires a very small amount of enzyme material in comparison with the method previously used.
