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Isolation of activating factors of serotonin N-acetyltransferase from rice peptides

Moritani, Chie, Kawakami, Kayoko, Fujita, Akiko, Kawakami, Koji, Shimoda, Hiroshi, Hatanaka, Tadashi, Tsuboi, Seiji
Journal of functional foods 2018 v.41 pp. 148-154
acetyltransferases, disulfide bonds, glutathione, hydrogen peroxide, mass spectrometry, melatonin, reversed-phase high performance liquid chromatography, rice, sequence analysis, serotonin
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.