Partial characterization of N-acetyltransferase activity from cerebral ganglia and malpighian tubules of Periplaneta americana

Martin, Richard J.; Jahagirdar, A.P.; Downer, Roger

doi:10.1016/0020-1790(89)90039-5

Cited by 20 publications

(8 citation statements)

References 24 publications

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“…Sci. USA 93 (1996) one aaNAT enzyme may be expressed in insects as previously 7~---l suggested (14,16). The fact that the gene encoding a dopamine acetyltransferase activity (5) and the aaNAT1 gene described here both were localized to the 60B locus on chromosome 2 suggests that either aaNAT1 exclusively encodes a dopaminẽ .…”

Section: Resultssupporting

confidence: 59%

“…However, it is not known if aaNAT activity and melatonin are subject to diurnal variation in Drosophila melanogaster. aaNAT activity has been described in several insect species (14)(15)(16)(17)(18), including D. melanogaster (2,19), but none of these enzymes could be purified to derive antibodies or sufficient internal sequence information for cloning. We have purified an aaNAT from D. melanogaster using tryptamine acetylation as a functional enzyme assay (20).…”

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confidence: 99%

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Cloning of an arylalkylamine N-acetyltransferase (aaNAT1) from Drosophila melanogaster expressed in the nervous system and the gut.

Hintermann

Grieder

Amherd

et al. 1996

Proc. Natl. Acad. Sci. U.S.A.

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In insects, neurotransmitter catabolism, melatonin precursor formation, and sclerotization involve arylalkylamine N-acetyltransferase (aaNAT, EC 2.3.1.87) activity. It is not known if one or multiple aaNAT enzymes are responsible for these activities. We recently have purified an aaNAT from Drosophila melanogaster. Here, we report the cloning of the corresponding aaNAT cDNA (aaNAT1) that upon COS cell expression acetylates dopamine, tryptamine, and the immediate melatonin precursor serotonin. aaNAT1 represents a novel gene family unrelated to known acetyltransferases, except in two weakly conserved amino acid motifs. In situ hybridization studies of aaNAT1 mRNA in embryos reveal hybridization signals in the brain, the ventral cord, the gut, and probably in oenocytes, indicating a broad tissue distribution of aaNAT1 transcripts. Moreover, in day/ night studies we demonstrate a diurnal rhythm of melatonin concentration without a clear-cut change in aaNATI mRNA levels. The data suggest that tissue-specific regulation of aaNAT1 may be associated with different enzymatic functions and do not exclude the possibility of additional aaNAT genes.Amine acetylation in insects by the enzyme arylalkylamine N-acetyltransferase (aaNAT, EC 2.3.1.87) is involved in at least three different physiological functions: (i) It appears to be the major route of neurotransmitter catabolism (1-4), inactivating biogenic amines including tryptamine, tyramine, octopamine, serotonin, and norepinephrine. (ii) An acetylated amine, acetyldopamine, is subsequently oxidized to the respective quinone, which cross-links different proteins and/or chitin, resulting in stabilization and hardening (sclerotization) of the insect cuticle (5). (iii) Acetylation of serotonin plays an important role in the regulation of photoperiodically influenced physiological and behavioral processes (6). The major regulatory hormone is melatonin (7, 8), which is synthesized by the methylation of acetylserotonin. In vertebrates, melatonin is secreted periodically with high concentration at night, caused by a 10-to 100-fold nocturnal increase in aaNAT activity (8). The final steps of aaNAT activation are still unclear and presumably involve new RNA and protein synthesis (9-12). All living organisms are influenced by environmental factors such as light and dark and thus need mechanisms to coordinate their physiological processes in response to seasonal changes. Serotonin, aaNAT activity, and melatonin have indeed been found in several organs (eye, optic lobe, and brain) of various invertebrate species in which melatonin concentration also shows a rhythmic behavior (13). However, it is not known if aaNAT activity and melatonin are subject to diurnal variation in Drosophila melanogaster. aaNAT activity has been described in several insect species (14-18), including D. melanogaster (2, 19), but none of these enzymes could be purified to derive antibodies or sufficient internal sequence information for cloning. We have purified an aaNAT from D. melanogaster using tryptam...

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Section: Resultssupporting

confidence: 59%

mentioning

confidence: 99%

Cloning of an arylalkylamine N-acetyltransferase (aaNAT1) from Drosophila melanogaster expressed in the nervous system and the gut.

Hintermann

Grieder

Amherd

et al. 1996

Proc. Natl. Acad. Sci. U.S.A.

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“…Since all organisms are influenced by environmental factors, such as light and darkness, they need a mechanism to coordinate their physiological processes in response to seasonal changes. In various invertebrate species, serotonin, aaNAT activity and melatonin have indeed been found in several organs, including eyes, optic lobe and brain [5][6][7][8][9][10][11], supporting the idea that melatonin is an evolutionary highly conserved endogenous mediator of photoperiodic information [5]. In insects, besides melatonin formation (stud-*Corresponding author.…”

Section: Introductionmentioning

confidence: 88%

Isolation and characterization of an arylalkylamine N‐acetyltransferase from Drosophila melanogaster

1995

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The enzyme arylalkylamine N-acetyltransferase (aaNAT) catalyzes the rate-limiting step in melatonin formation in the vertebrate pineal gland. Numerous attempts to purify this highly unstable enzyme from vertebrates have been unsuccessful. Here, we report the purification of an aaNAT enzyme from Drosophila melanogaster, using a radioenzymatic activity assay and column chromatography. The isolated 29.5-kDa protein acetylates tryptamine, dopamine and serotonin with affinities of 0.89 to 0.97 mM, respectively. This suggests that the identified aaNAT may be involved in melatonin synthesis and sclerotization as well as in neurotransmitter catabolism in insects.

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“…Tissues were sonicated with 5 pulses at 50% duty and 20 W output to the microtip. Following centrifugation at 40,OOOg for 20 min at 4"C, the supernatants, which contained at least 90% of the NAT activity [13], were kept on ice for no more than 30 min prior to assay.…”

Section: Tissue Homogenization and Enzyme Assaymentioning

confidence: 99%

“…Assay conditions were optimized as described previously [13]. Briefly, tis- Figure 1A,B illustrates inhibition of NAT from Malpighian tubules and cerebral gangha, respectively, by sulfhydryl inhibitors.…”

Section: Tissue Homogenization and Enzyme Assaymentioning

confidence: 99%

Effects of potential inhibitors on N‐acetylation of octopamine by tissue extracts from malpighian tubules and cerebral ganglia of Periplaneta americana L

Martin

Downer

1989

Arch Insect Biochem Physiol

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N-acetyltransferase activity in extracts of Malpighian tubules and cerebral ganglia from Periplaneta americana L. was monitored using high performance liquid chromatography with coulometric electrochemical detection. Several potential inhibitors were tested against the tissue preparations, and the results indicate distinct differences in the nature of the enzyme derived from the two sources. The Malpighian tubule and cerebral ganglia extracts were sensitive to sulfhydryl inhibition and divalent cations, and both preparations also showed end-product inhibition with coenzyme A. Juglone, an inhibitor of choline acetyltransferase, inhibits N-acetyltransferase activity from both tissues; however, separate studies with choline indicate that the N-acetylation observed in this study is not due to choline acetyltransferase. A number of phenyl, phenol, catechol and indole derivatives were tested and the results indicate that the presence of a 4-hydroxyl group on the phenol ring enhances the capacity of phenol and catechol derivatives to inhibit N-acetyltransferase. Demethylchlordimeform inhibits the production of N-acetyl-p-Octopamine by Malpighian tubule preparations and intact tissues whereas other formamidines, BTS-27271 and amitraz, inhibit N-acetyl-p-Octopamine production only in intact tissues.

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Partial characterization of N-acetyltransferase activity from cerebral ganglia and malpighian tubules of Periplaneta americana

Cited by 20 publications

References 24 publications

Cloning of an arylalkylamine N-acetyltransferase (aaNAT1) from Drosophila melanogaster expressed in the nervous system and the gut.

Cloning of an arylalkylamine N-acetyltransferase (aaNAT1) from Drosophila melanogaster expressed in the nervous system and the gut.

Isolation and characterization of an arylalkylamine N‐acetyltransferase from Drosophila melanogaster

Effects of potential inhibitors on N‐acetylation of octopamine by tissue extracts from malpighian tubules and cerebral ganglia of Periplaneta americana L

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