Determination of kinetic properties of serotonin-N-acetyltransferase in bovine pineal gland using HPLC with fluorimetric detection.
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.
Fajardo
N
N
Departamento de Fisiología, Facultad de Medicina, Universidad de La Laguna, Tenerife, Spain.
Abreu
P
P
Alonso
R
R
D000818:Animals; D001191:Arylamine N-Acetyltransferase / Q000378:metabolism*; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003623:Dark Adaptation; D005470:Fluorometry; D006243:Harderian Gland / Q000201:enzymology; D007700:Kinetics; D008297:Male; D009924:Organ Culture Techniques; D010870:Pineal Gland / Q000201:enzymology*; D051381:Rats; D012160:Retina / Q000201:enzymology
D001191:Arylamine N-Acetyltransferase