Purification and characterization of arylalkylamine N-acetyltransferase from cockroach testicular organs

Arylalkylamine N-acetyltransferase (NAT; EC2.3.1.87) catalyzes N-acetylation of various arylalkylamines using acetyl-CoA as a donor substrate. A type of NAT was purified 2700-fold from 451 pairs of cockroach testicular organs consisting of testis and its accessory gland. The NAT activity was recovered as a single peak on any column chromatography examined, suggesting that the testicular organ contained only one form of NAT. Five steps of successive column chromatographies gave a single protein band on SDS-polyacrylamide gel electrophoresis with estimated molecular mass of 28 kDa. The molecular mass of the native enzyme was also determined to be approximately 30 kDa by molecular sieve chromatography, indicating that the enzyme is a monomer protein. The enzyme acted on various arylalkylamines such as tryptamine, serotonin, dopamine, octopamine, norepinephrine, tyramine and methoxytryptamine, with K(m) values ranging from 20 to 50 microM. The optimum pH for these substrates was around 6.0. Internal amino acid sequences derived from two proteolytic fragments of the enzyme were determined as Leu-Leu-Gly-Glu-Asn-Gly-Asp-Glu and Phe-Phe-Phe-Leu-Glu-Glu-Pro-Leu-Asn-Ile-Ser-Leu-Gln, both of which exhibited significant homology to the C-terminal sequence of known vertebrate NATs; however, homology was less than 45%. These results suggest that a unique NAT is present in the cockroach testicular organ at high levels, and likely plays a role in the regulation of testicular function.