Histamine /V-methyltransferase catalyzes the transfer of a methyl group from S-adenosyl-L-methionine to histamine to form N-methylhistamine. This assay is suitable for following activity during enzyme purification.
Histamine and N-methylhistamine were separated on a weak cation exchange (TSK CM2SW) column (4 mm X 150 mm). The mobile phase was prepared by dissolving 5.25 g of citric acid and 10 g of imidazole in 880 mL of water, and then mixing the resulting solution with 200 mL of acetonitrile. The separated compounds were derivatized at room temperature with o-phthaldialdehyde by mixing that reagent with the column eluate at a flow rate of 1.0 mL/min. The o-phthaldialdehyde reagent was prepared by dissolving 2.47 g of boric acid and 0.12 g of Brij 35 in 100 mL water, adjusting the pH to 10.5 with 5 M KOH, and then adding 60 mg of o-phthaldialdehyde in 5 mL of methanol together with 100 /aL of 2-mercaptoethanol. The reaction coil was made of Teflon tubing (1 m x 0.5 mm), and the resulting products were detected by fluorescence. The ranges of excitation and emission wavelengths were 230 to 400 and 410 to 800 nm, respectively.
The reaction mixture in a final volume of 1 mL contained 0.7 mL of 0.1 M phosphate buffer (pH 7.4) containing 0.1 mM pargyline and 0.1 mM aminoguanidine, 0.1 mL of 1.0 mM histamine, 0.1 mL of 2.5 mM S-adenosyl-L-methionine, and 0.1 mL of enzyme preparation. After incubation at 37°C, the reaction was terminated by adding 50 pL of 60% perchloric acid. After centrifugation, the supernate was adjusted to pH 6.5 with 5 M KOH after adding 20 ju,L of 5% bromothymol blue as a pH indicator. The precipitated potassium Perchlorate was removed by centrifugation and the supernate was applied to a column of Amberlite CG-50 (4 mm x 20 mm), which had been equilibrated with 0.2 M sodium phosphate buffer (pH 6.5). The column was washed with 3 mL of 5 mM Na2EDTA (pH 6.5) and with 3 mL of 0.1 M hydrochloric acid. Histamine and JV-methylhistamine were eluted with 1.0 mL of 0.5 M HCl, and the eluate was applied to the HPLC column.
The source of enzyme was a dialyzed, high speed supernate obtained after fresh rat kidney had been homogenized in 4 volumes of ice-cold 0.05 M sodium phosphate buffer (pH 7.4) containing 1 mM dithiothreitol and 1% polyethylene glycol (average molecular mass 300).
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