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. K(m) and V(max) estimations for acetyl-CoA at a 10 mM tryptamine concentration were 63.3-mu-M and 4.42 nmol/h per eye, respectively. Regardless of the acceptor amine (tryptamine or serotonin), the K(m) was not affected by the acetyl-CoA concentration (50 or 250-mu-M), whereas the V(max) was significantly increased at a 250-mu-M acetyl-CoA concentration. Ocular NAT showed a higher affinity for serotonin (K(m) = 20.7-mu-M) than for tryptamine (K(m) = 48-60-mu-M); V(max), 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.