The N-acetylation of different aryl- and arylalkylamines, using acetyl-CoA as the acetyl donor, proceeds when catalyzed by acetyl-Co A: arylamine N-acetyltransferase. A natural substrate is serotonin, which is converted to N-acetylserotonin in the pineal gland and other regions of the brain.
Serotonin and /V-acetylserotonin are separated on a Spherisorb ODS 2 column (3.2 mm X 150 mm). The mobile phase was a 3:1 mixture of 0.1 M sodium acetate (pH 4.75) and methanol. The column effluent was monitored at 275 nm. Greater sensitivity can be obtained by using fluorescence or electrochemical detection.
The enzyme assay mixture contained in a final volume of 200 fiL: 20 pimol glycine-KOH buffer (pH 9.5), 0.30 /xmol serotonin-HCl, 0.12 /xmol acetyl-CoA, and 50 jaL of enzyme solution with a maximum activity of 100 mU/mL. After 5 minutes of incubation at 35°C, the reaction was stopped by diluting 10-fold with 0.1 M perchloric acid. After filtering, a 25 /xL aliquot of the filtrate was injected onto the HPLC column.
Acetyl-CoA: arylamine /V-acetyltransferase was purified from pigeon liver by using steps involving protamine sulfate, ammonium sulfate fractionation, and affinity chromatography on immobilized amethopterin. Figure 9.20 shows a representative chromatogram. In another study, Thomas et al. (1990) used tryptamine as the substrate. In addition, whereas the study by Manneus et al. (1990) appeared to require pure enzyme, Thomas's study did not.
Was this article helpful?