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首页> 外文期刊>DNA and Cell Biology >Purification, Cloning, and Characterization of a Second Arylalkylamine N-Acetyltransferase from Drosophila melanogaster
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Purification, Cloning, and Characterization of a Second Arylalkylamine N-Acetyltransferase from Drosophila melanogaster

机译：果蝇的第二个芳烷基胺N-乙酰基转移酶的纯化，克隆和表征

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摘要

In insects, amine acetylation by the enzyme arylalkylamine N-acetyltransferase (AANAT) is involved in melatonin formation, sclerotization, and neurotransmitter inactivation. This wide spectrum of activities suggests that several AANAT enzymes are present. We recently purified a protein fraction with AANAT activity from Drosophila melanogaster and cloned the corresponding gene, aaNAT1. Following the same strategy, we now report the purification of an additional AANAT from D. melanogaster, AANAT2, and the cloning of the corresponding cDNA. The isolated protein differs from AANAT1a and AANAT1b in its molecular weight and isoelectric point. The AANAT2 shares about 30% identity with the products of the aaNAT1 gene. The enzyme does not follow one-site Michaelis-Menten kinetics when assayed with various concentrations of the arylalkylamine tryptamine and a constant concentration (0.5 mM) of the cofactor acetyl coenzyme A. The data can be interpreted in terms of an enzyme with two kinetic regimes (Km1 = 7.2 μM, Km2 = 0.6 mM, and vmax2 = 2.7 vmax1) that are governed by binding of the substrate to a regulatory site (Kr = 6.2 mM). These findings demonstrate the presence of a second expressed gene encoding an AANAT in D. melanogaster. Northern blot analysis revealed no diurnal variation of aaNAT2 transcription, similar to the results obtained for aaNAT1a and aaNAT1b.
机译：在昆虫中，芳基烷基胺N-乙酰基转移酶（AANAT）引起的胺乙酰化与褪黑激素的形成，硬化和神经递质失活有关。这种广泛的活性表明存在几种AANAT酶。我们最近从果蝇中纯化了具有AANAT活性的蛋白质级分，并克隆了相应的基因aaNAT1。遵循相同的策略，我们现在报道从D. melanogaster纯化另外的AANAT，AANAT2，并克隆相应的cDNA。分离的蛋白质在分子量和等电点上与AANAT1a和AANAT1b不同。 AANAT2与aaNAT1基因的产物具有大约30％的同一性。当使用各种浓度的芳基烷基胺类色胺和恒定浓度（0.5 mM）的辅因子乙酰辅酶A进行测定时，该酶不遵循单点Michaelis-Menten动力学。该数据可以用两种动力学方案的酶来解释（Km1 = 7.2μM，Km2 = 0.6 mM，vmax2 = 2.7 vmax1）由底物与调节位点的结合（Kr = 6.2 mM）控制。这些发现证明在黑腹果蝇中存在第二个编码AANAT的表达基因。 Northern印迹分析显示，aaNAT2转录没有昼夜变化，与获得aaaNAT1a和aaNAT1b的结果相似。

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