Determination of kinetic properties of serotonin‐N‐acetyltransferase in bovine pineal gland using HPLC with fluorimetric detection

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Abstract
The determination of serotonin-N-acetyltransferase (NAT) activity in the bovine pineal gland and other rat tissues was based upon the separation and detection of N-acetyltryptamine formed from tryptamine and acetyl CoA by means of high performance liquid chromatography with fluorimetric detection. In the bovine pineal the enzyme exhibited a Km value of 31.45 +/- 4.98 microM and a Vmax value of 30.90 +/- 1.18 pmol N-acetyltryptamine/min/mg protein for tryptamine, and a Km value of 28.72 +/- 7.50 microM and a Vmax value of 25.90 +/- 1.50 pmol N-acetyltryptamine/min/mg protein for acetyl CoA. The present method is simple, allows the determination of NAT activity from a variety of enzyme sources, has application to pharmacological studies of NAT regulation in tissue cultures, and provides an alternative to current radioenzymatic assays.