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Isolation of activating factors of serotonin N-acetyltransferase from rice peptides
Journal of Functional Foods ( IF 5.6 ) Pub Date : 2017-12-26 , DOI: 10.1016/j.jff.2017.12.051
Chie Moritani , Kayoko Kawakami , Akiko Fujita , Koji Kawakami , Hiroshi Shimoda , Tadashi Hatanaka , Seiji Tsuboi

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.



中文翻译:

大米肽中5-羟色胺N-乙酰基转移酶激活因子的分离

血清素Ñ -acetyltransferase(AA-NAT),它转换血清素到Ñ -acetylserotonin,为褪黑激素合成的限速酶。我们先前曾报道细胞内氧化还原条件通过以谷胱甘肽(GSH)依赖的方式切换分子内二硫键来调节AA-NAT活性。因此,增加的谷胱甘肽水平可以激活AA-NAT。在这里,我们发现市售的大米肽可增加细胞内GSH含量并减弱H 2 O 2。诱导组成型表达AA-NAT的COS7细胞中AA-NAT活性的失活。使用Sep-Pak C18柱和三轮反相HPLC纯化肽,然后通过质谱进行序列分析,从而鉴定出了三种肽。这些肽中的两种(Pep2,VVTFGPSGLTTEVK; Pep3,YQQQFQQFLPEGQSQSQK)阻止了H 2 O 2灭活AA-NAT活性。只有Pep3可以增加细胞内GSH的水平,并防止H 2 O 2诱导的还原/氧化GSH比率的降低。这些结果表明,Pep3通过增加细胞内GSH水平来激活AA-NAT,从而改变AA-NAT的分子内二硫键。

更新日期:2017-12-26
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