ABSTRACT
(+)-S-Adenosyl-L-methionine [(+)-SAM] was isolated from rat brain and was quantified by HPLC followed by UV spectrophotometric measurements and by 1H-NMR. Its estimated ratio in brain is 3% of total SAM. Because of its commercial unavailability, (+)-SAM was also prepared from chemically synthesized SAM by separation of the two diastereoisomers on a preparative reverse-phase Nucleosil C8 column. The (+) diastereoisomer thus obtained was then assayed in vitro both as an inhibitor and a substrate of phenylethanolamine N-methyltransferase. Enzymatic activity was measured by HPLC analysis. It was shown that (+)-SAM has no effect on phenylethanolamine N-methyltransferase activity; therefore, it is unlikely that (+)-SAM plays any possible role in regulation of adrenaline synthesis in the brain.
Subject(s)
Brain/metabolism , Phenylethanolamine N-Methyltransferase/metabolism , S-Adenosylmethionine/metabolism , Animals , Chromatography, High Pressure Liquid , Kinetics , Magnetic Resonance Spectroscopy , Male , Rats , Rats, Sprague-Dawley , S-Adenosylmethionine/chemistry , S-Adenosylmethionine/pharmacology , Stereoisomerism , Ultraviolet RaysABSTRACT
A simple and rapid method for measuring phenylethanolamine N-methyltransferase (PNMT) activity by high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection is described. This assay requires a partially purified PNMT preparation derived from bovine adrenals, with noradrenaline and S-adenosyl-L-methionine (SAM) as co-substrates. After incubation, the reaction is stopped by addition of acid and the reaction mixture is analysed directly by HPLC. The enzymatically formed S-adenosyl-L-homocysteine (SAH) is detected at 258 nm and determined. Under optimum conditions, the stability of SAH allowed automation of the HPLC detection. This assay was validated by the determination of the kinetic properties of PNMT. Km values for noradrenaline and SAM defined in this assay (16 and 5.7 microM, respectively) are consistent with previously published values. This assay is simple enough to be used for large series of measurements of PNMT activity testing new methyl acceptors, potential inhibitors or PNMT activity in adrenal medulla.
