HYDROXYINDOLE-O-METHYLTRANSFERASE ACTIVITY ASSAY USING HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY WITH FLUOROMETRIC DETECTION - DETERMINATION OF MELATONIN ENZYMATICALLY FORMED FROM N-ACETYLSEROTONIN AND S-ADENOSYL-L-METHIONINE

A reliable, sensitive and rapid assay has been developed for determini ng the activity of hydroxyindole-O-methyltransferase (HIOMT; L-methion ine:N-acetylserotonin-O-methyltransferase EC 2.1.1.4), which catalyzes the final step in the melatonin (N-acetyl-5-methoxytryptamine) biosyn thetic pathway. This method is based on the separation and detection o f melatonin formed enzymatically from N-acetylserotonin and S-adenosyI -L-methionine, by high-performance liquid chromatography with fluorome tric detection. The detection limit far melatonin formed per sample wa s as low as 150 fmol, indicating that the sensitivity of this assay wa s comparable to that of a radioisotopic assay. The assay was applied t o the determination of HIOMT activity in rat pineal gland. The HIOMT a ctivity obtained in this study was comparable with, or slightly lower than those reported previously using radioisotopic assays.