Arylalkylamine (serotonin) N-acetyltransferase assay using high-performance liquid chromatography with fluorescence or electrochemical detection of N-acetyltryptamine

doi:10.1016/0003-2697(90)90673-W

Analytical Biochemistry

Volume 184, Issue 2, 1 February 1990, Pages 228-234

https://doi.org/10.1016/0003-2697(90)90673-W Get rights and content

Abstract

A sensitive, rapid, and economical method has been developed for determination of serotonin N-acetyltransferase activity from a variety of enzyme sources. The assay is based upon separation and detection of N-acetyltryptamine formed from tryptamine and acetyl coenzyme A, by means of high-performance liquid chromatography with either electrochemical or fluorometric detection. The limit of sensitivity with both detection methods is less than 20 pmol of N-acetyltryptamine formed per sample. A method for synthesis of N-acetyltryptamine, used as an external standard in the assay, is described.

References (14)

S. Binkley
Pineal and Retinal Relationship
A.F. Wiechmann
Exp. Eye Res
(1986)
T. Deguchi et al.
Anal. Biochem
(1972)
O.H. Lowry et al.
J. Biol. Chem
(1951)
L. Miller et al.
Comp. Biochem. Physiol. C
(1980)
P. Voisin et al.
J. Biol. Chem
(1984)
D.C. Klein et al.
Science
(1970)

There are more references available in the full text version of this article.

Cited by (51)

Isolation of activating factors of serotonin N-acetyltransferase from rice peptides
2018, Journal of Functional Foods
Citation Excerpt :
The sum of reduced GSH and GSSG was expressed as “total GSH”. AA-NAT activity was assayed as described previously (Thomas, Zawilska, & Iuvone, 1990; Tsuboi et al., 2001). COS7/NAT cells were seeded into six-well plates at a concentration of 1.5 × 105 cells/well.
Serotonin N-acetyltransferase (AA-NAT), which converts serotonin to N-acetylserotonin, is a rate-limiting enzyme for melatonin synthesis. We previously reported that intracellular redox conditions regulate AA-NAT activity by switching an intramolecular disulfide bridge in a glutathione (GSH)-dependent manner; thus, increased GSH levels can activate AA-NAT. Here, we found that commercially available rice peptides increased intracellular GSH levels and attenuated H₂O₂-induced inactivation of AA-NAT activity in COS7 cells expressing AA-NAT constitutively. Purification of the peptides using a Sep-Pak C18 cartridge and three rounds of reversed-phase HPLC, followed by sequence analysis by mass spectrometry, led to the identification of three peptides. Two of these peptides (Pep2, VVTFGPSGLTTEVK; Pep3, YQQQFQQFLPEGQSQSQK) prevented inactivation of AA-NAT activity by H₂O₂. Only Pep3 increased intracellular GSH levels and prevented the decrease in the reduced/oxidised GSH ratio induced by H₂O₂. These results suggest that Pep3 activates AA-NAT by increasing intracellular GSH levels, which switches the intramolecular disulfide bond of AA-NAT.
Serotonin metabolism in rat skin: Characterization by liquid chromatography-mass spectrometry
2004, Archives of Biochemistry and Biophysics
We have recently uncovered the full expression of novel cutaneous serotoninergic and melatoninergic systems in the human and hamster skin. In this work, we have characterized serotonin metabolism in the rat skin using liquid chromatography–mass spectrometry and found that serotonin undergoes acetylation in the presence of acetyl coenzyme A. Inhibition of serotonin acetylation with Cole bisubstrate inhibitor shows that rat skin expresses both arylalkylamine and arylamine N-acetyltransferase activities. The serotonin degradation product—5-hydroxyindole acetic acid is also detected and pargyline (monoaminooxidase inhibitor) suppresses almost completely 5-hydroxyindole acetic acid accumulation. Together with previous data, the present study clearly demonstrates that biotransformation of serotonin in mammalian skin follows two alternate pathways. In the first pathway, serotonin is acetylated by arylalkylamine and arylamine N-acetyltransferases to generate the precursor of melatonin. Alternately, serotonin may undergo oxidative deamination by monoaminooxidase followed by enzymatic degradation by aldehyde dehydrogenase into 5-hydroxyindole acetic acid, which is presumably devoid of biological activity. Thus, the current methodological development of a liquid chromatography–mass spectrometry-based assay allows rapid resolution of the cutaneous metabolism of serotonin.
Circadian rhythm and photic control of cAMP level in chick retinal cell cultures: A mechanism for coupling the circadian oscillator to the melatonin-synthesizing enzyme, arylalkylamine N-acetyltransferase, in photoreceptor cells
2003, Brain Research
Citation Excerpt :
Cells for AANAT measurements were collected in 150 μl of 0.25 M potassium phosphate (pH 6.5) containing 1.33 mM acetylcoenzyme A. Samples were frozen on dry ice and kept at −80 °C until analyzed. The AANAT enzyme activity was determined in cell homogenates by measuring the catalytic formation of N-acetyltryptamine from tryptamine and acetylcoenzyme A, as described in Ref. [52]. The reaction product, N-acetyltryptamine, was quantified by HPLC with fluorescence detection.
Arylalkylamine N-acetyltransferase (AANAT) is the penultimate and key regulatory enzyme in the melatonin biosynthetic pathway. In chicken retina in vivo, AANAT is expressed in a circadian fashion, primarily in photoreceptor cells. AANAT activity is high at night in darkness, low during the daytime, and suppressed by light exposure at night. In the present study, we investigated the circadian and photic regulation of adenosine 3′,5′-monophosphate (cAMP) in cultured retinal cells entrained to a daily light–dark (LD) cycle, as well as the role of Ca²⁺ and cAMP in the regulation of AANAT activity. Similar to AANAT activity, cAMP levels fluctuate in a daily fashion, with high levels at night in darkness and low levels during the day in light. This daily fluctuation continued with reduced amplitude in constant (24 h/day) darkness (DD). These changes in cAMP appear to be causally related to control of AANAT activity. Adenylyl cyclase and protein kinase A inhibitors suppress the noctural increase of AANAT in DD, while 8Br-cAMP augments it. The nocturnal increase of AANAT activity also involves Ca²⁺ influx, as it is inhibited by nitrendipine, an inhibitor of L-type voltage-gated channels, and augmented by Bay K 8644, a Ca²⁺ channel agonist. The effect of Bay K 8644 was antagonized by the adenylyl cyclase inhibitor MDL 12330A, suggesting a link between Ca²⁺ influx, cAMP formation, and AANAT activity in retinal cells. Light exposure at night, which rapidly suppresses AANAT activity, also suppressed cAMP levels. The effect of light on AANAT activity was reversed by Bay K 8644, 8Br-cAMP, and the proteasome inhibitor lactacystin. These results indicate a dynamic interplay of circadian oscillators and light in the regulation of cAMP levels and AANAT activity in photoreceptor cells.
Melatonin synthesis in retina: Circadian regulation of arylalkylamine N-acetyltransferase activity in cultured photoreceptor cells of embryonic chicken retina
2003, Brain Research
Citation Excerpt :
Cells for AANAT measurements were collected in 150 μl of 0.25 M potassium phosphate (pH 6.5) containing 1.12 mM acetylcoenzyme A. Samples were frozen in dry ice and kept at −80 °C until analyzed. The AANAT enzyme activity was determined in cell homogenates by measuring the catalytic formation of N-acetyltryptamine from tryptamine and acetylcoenzyme A, as described by Thomas et al. [25]. The reaction product, N-acetyltryptamine, was quantified by HPLC with fluorescence detection.
The key regulatory enzyme in melatonin synthesis is arylalkylamine N-acetyltransferase (AANAT). In vivo, AANAT activity in chicken retinal photoreceptor cells exhibits a circadian rhythm that peaks at night. The purpose of the present study was to investigate the temporal development of light/dark and circadian oscillations of AANAT activity in cultured retinal cells prepared from 6- and 8-day-old chicken embryos (E6, E8, respectively). Photoreceptor cells prepared from E6 retinas and incubated under a 14-h light/10-h dark (LD) cycle of illumination for 5–7 days displayed prominent daily fluctuations in AANAT activity on days 5 and 6 in vitro. However, when E6 cells, incubated for 5 days under LD, were transferred to continuous (24 h/day) darkness (DD) on day 6, no daily pattern of activity was observed. This result indicates that AANAT fluctuations were light-driven and not circadian at this stage. In contrast, cells prepared from E8 embryos and incubated under conditions identical to those for E6 cells displayed prominent rhythms of AANAT activity in both LD and DD, indicative of circadian control. To determine if circadian control of AANAT activity would develop in E6 cells incubated for a longer period of time to allow maturation, cells were incubated for 8 days in LD followed by 2 days in DD. AANAT activity in these cells was rhythmic in both LD and DD. In cells incubated in this manner, a 2-h light pulse in the middle of the subjective night suppressed AANAT activity, indicating that the enzyme activity in the cultured cells is acutely suppressed by light, as it is in vivo. These results indicate that the ability to express circadian regulation of AANAT activity is an intrinsic property of retinal cells that can develop in vitro. Development of light–dark regulation of AANAT activity appears to precede the circadian clock-control of enzyme activity.
Voltammetric and flow amperometric methods for the determination of melatonin in pharmaceuticals
2003, Journal of Pharmaceutical and Biomedical Analysis
Melatonin can be sensitively detected in pharmaceuticals by two different and simple electrochemical methods: cyclic voltammetry (CV) and amperometric detection in a flow injection analysis system (FIA-ED). An adequate pre-treatment of the carbon paste electrode in the first case and the employ of a high flow rate in the second one were the key for obtaining a very good reproducibility (R.S.D. values of 1.5 (n=10) and 1.3% (n=20), respectively). Low limits of detection were achieved and with the coupling of a flow system a linear dynamic range of three orders of magnitude (from 10⁻⁸ to 10⁻⁵ M) was obtained. Both methods were applied to the determination of melatonin in pharmaceuticals. In order to best validate these methodologies a fluorescent procedure was developed to contrast the results. As no interferences from the matrix were found the employ of a separation technique is not necessary. In this way the procedure is fastened and simplified. Moreover, the low price, ease of handling, possibility of automation and high sample throughput are important advantages that convert the flow methodology in an attractive alternative for quality control of pharmaceuticals.
An intramolecular disulfide bridge as a catalytic switch for serotonin N-acetyltransferase
2002, Journal of Biological Chemistry
Citation Excerpt :
The fractions containing AA-NAT activity were pooled and stored at −85 °C until use. AA-NAT activity was assayed according to the previously published procedure (27). Briefly, the purified enzyme (5 μg protein/assay) was added to an assay mixture comprising 0.25 m potassium phosphate buffer (pH 7.5) containing 1.5 mm acetyl-CoA and 2 mmtryptamine.
Serotonin N-acetyltransferase (EC. 2.3.1.87) (AA-NAT) is a melatonin rhythm-generating enzyme in pineal glands. To establish a melatonin rhythm, AA-NAT activity is precisely regulated through several signaling pathways. Here we show novel regulation of AA-NAT activity, in which an intramolecular disulfide bond may function as a switch for the catalysis. Recombinant AA-NAT activity was irreversibly inhibited byN-ethylmaleimide (NEM) in an acetyl-CoA-protected manner. Oxidized glutathione or dissolved oxygen reversibly inhibited AA-NAT in an acetyl-CoA-protected manner. To identify the cysteine residues responsible for the inhibition, AA-NAT was first oxidized with dissolved oxygen, treated with NEM, reduced with dithiothreitol, and then labeled with [¹⁴C]NEM. Cys⁶¹ and Cys¹⁷⁷ were specifically labeled in an acetyl-CoA-protected manner. The AA-NAT with the Cys⁶¹to Ala and Cys¹⁷⁷ to Ala double substitutions (C61A/C177A-AA-NAT) was fully active but did not exhibit sensitivity to either oxidation or NEM, whereas the AA-NATs with only the single substitutions retained about 40% of these sensitivities. An intramolecular disulfide bond between Cys⁶¹ and Cys¹⁷⁷ formed upon oxidation and cleaved upon reduction was identified. Furthermore, C61A/C177A-AA-NAT expressed in COS7 cells was relatively insensitive to H₂O₂-evoked oxidative stress, whereas wild-type AA-NAT was strongly inhibited under the same conditions. These results indicate that the formation and cleavage of the disulfide bond between Cys⁶¹ and Cys¹⁷⁷ produce the active and inactive states of AA-NAT. It is possible that intracellular redox conditions regulate AA-NAT activity through switching via an intramolecular disulfide bridge.

View all citing articles on Scopus

¹: Present address: Department of Pharmacodynamics, Medical Academy of Lodz, Lodz 90-151, Poland.

View full text

Arylalkylamine (serotonin) N-acetyltransferase assay using high-performance liquid chromatography with fluorescence or electrochemical detection of N-acetyltryptamine

Abstract

Exp. Eye Res

Anal. Biochem

J. Biol. Chem

Comp. Biochem. Physiol. C

J. Biol. Chem

Science