Characterization of serotonin N-acetyltransferase in the lateral eye of the green frog Rana perezi: protective action of EGTA.

The kinetics of serotonin N-acetyltransferase (NAT) from the lateral eye of Rana perezi have been characterized. NAT from ocular tissue reached maximal activity at a phosphate buffer concentration of 250 mM and a pH of 6.5. Reaction linearity was highly conserved within the homogenate fraction range tested (0.033-0.33). The time course of ocular NAT reaction showed a high linearity at 25 and 35 degrees C. Km and Vmax estimations for acetyl-CoA at a 10 mM tryptamine concentration were 63.3 microM and 4.42 nmol/h per eye, respectively. Regardless of the acceptor amine (tryptamine or serotonin), the Km was not affected by the acetyl-CoA concentration (50 or 250 microM), whereas the Vmax was significantly increased at a 250 microM acetyl-CoA concentration. Ocular NAT showed a higher affinity for serotonin (Km = 20.7 microM) than for tryptamine (Km = 48-60 microM); Vmax, however, was similar for both substrates. Acetyl-CoA does not protect ocular NAT; in contrast, the use of EGTA (greater than or equal to 4 mM) in the assay is essential to protect the enzyme because NAT in ocular crude homogenate shows rapid inactivation. This result suggests that intracellular calcium levels are involved in the NAT inactivation mechanisms in frog ocular tissue.