Assay of phenylethanolamine N-methyltransferase activity using high-performance liquid chromatography with ultraviolet absorbance detection.
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.
Beaudouin
C
C
Laboratoire de Neuropharmacologie-CNRS UMR 105, Faculté de Pharmacie, Université Claude Bernard, Lyon, France.
Haurat
G
G
Fraisse
L
L
Souppe
J
J
Renaud
B
B
D000311:Adrenal Glands / Q000201:enzymology; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D007700:Kinetics; D008297:Male; D009638:Norepinephrine; D010625:Phenylethanolamine N-Methyltransferase / Q000032:analysis*; D051381:Rats; D012436:S-Adenosylmethionine / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity
D012436:S-Adenosylmethionine; D010625:Phenylethanolamine N-Methyltransferase; D009638:Norepinephrine