Characterization of indolamine N-acetyltransferase activity from the head ganglia of the American cockroach, Periplaneta americana

Abstract

More than 90% of indolamine N-acetyltransferase (NAT) activity was recovered in the supernatant of centrifuged homogenate of the head ganglia of the American cockroach, Periplaneta americana. NAT activity increased at temperatures of up to 40°C with serotonin (5HT) and up to 45°C with tryptamine (TP) as the substrates. The pH-activity curve with 5HT resembled that with TP, both showing two activity maxima, one at pH 7.0 and the other at 8.5-9.0. Na⁺ and K⁺ ions stimulated the enzymatic activity up to 200 mM, after which a further increase in the concentrations reduced the enzyme activity. Cu²⁺ suppressed the activity at least up to 0.5 mM. One millimolar Mg²⁺ stimulated the activity but a further increase in the concentration suppressed the activity. Ca²⁺ also stimulated the activity up to 2 mM, but a further increase suppressed the activity. The secondary plot of the ordinate intercepts of a series of Lineweaver-Burk plots against fixed concentrations of acetyl-CoA showed that the enzymatic mechanism of indolamine NAT from the head ganglia of P. americana proceeds according to the kinetics known as the ping pong Bi-Bi mechanism. The apparent K_ms and V_max with TP and acetyl-CoA were 19.3 μM and V_max 3.64 nmol product/mg protein/min, respectively, while the K_m for acetyl-CoA was 28.0 μM and V_max the same as that for TP. The K_m for 5HT was 106 μM, V_max being 2.16 nmol product/mg protein/min, while the K_m for acetyl-CoA was 19.7 μM, V_max being the same as that for 5HT.