Chapman KD, Venables B, Markovic R, Bettinger C
Department of Biological Sciences, Division of Biochemistry and Molecular Biology, University of North Texas, Denton, Texas 76203-5220 (K.D.C., R.M., R.W.B., C.B.).
Plant Physiol. 1999 Aug;120(4):1157-64. doi: 10.1104/pp.120.4.1157.
N-Acylethanolamines (NAEs) were quantified in seeds of several plant species and several cultivated varieties of a single species (cotton [Gossypium hirstutum]) by gas chromatography-mass spectroscopy. The total NAE content of dry seeds ranged from 490 +/- 89 ng g(-1) fresh weight in pea (Pisum sativum cv early Alaska) to 1,608 +/- 309 ng g(-1) fresh weight in cotton (cv Stoneville 7A glandless). Molecular species of NAEs in all seeds contained predominantly 16C and 18C fatty acids, with N-linoleoylethanolamine (NAE18:2) being the most abundant (approaching 1,000 ng g(-1) fresh weight in cottonseeds). Total NAE levels dropped drastically following 4 h of imbibition in seeds of pea, cotton, and peanut (Arachis hypogea cv Virginia), and this decline was most pronounced for NAE18:2. A novel enzyme activity was identified in cytosolic fractions of imbibed cottonseeds that hydrolyzed NAE18:2 in vitro. NAE degradation was optimal at 35 degrees C in 50 mM MES buffer, pH 6.5, and was inhibited by phenylmethylsulfonyl fluoride and 5, 5'-dithio-bis(2-nitrobenzoic acid), which is typical of other amide hydrolases. Amidohydrolase activity in cytosolic fractions exhibited saturation kinetics toward the NAE18:2 substrate, with an apparent K(m) of 65 &mgr;M and a V(max) of 83 nmol min(-1) mg(-1) protein. Total NAE amidohydrolase activity increased during seed imbibition, with the highest levels (about four times that in dry seeds) measured 2 h after commencing hydration. NAEs belong to the family of "endocannabinoids," which have been identified as potent lipid mediators in other types of eukaryotic cells. This raises the possibility that their imbibition-induced metabolism in plants is involved in the regulation of seed germination.
通过气相色谱 - 质谱联用技术对几种植物种子以及单一物种（棉花[陆地棉]）的多个栽培品种种子中的N - 酰基乙醇胺（NAEs）进行了定量分析。干种子中NAEs的总含量范围为：豌豆（早熟阿拉斯加豌豆品种）鲜重490±89 ng g⁻¹至棉花（斯通维尔7A无腺体品种）鲜重1,608±309 ng g⁻¹。所有种子中NAEs的分子种类主要含有16碳和18碳脂肪酸，其中N - 亚油酰乙醇胺（NAE18:2）含量最为丰富（棉籽中接近1,000 ng g⁻¹鲜重）。在豌豆、棉花和花生（弗吉尼亚花生品种）种子吸胀4小时后，NAEs的总水平急剧下降，其中NAE18:2的下降最为明显。在吸胀的棉籽胞质组分中鉴定出一种新的酶活性，该酶在体外可水解NAE18:2。NAE降解在50 mM MES缓冲液（pH 6.5）中35℃时最适宜，并且受到苯甲基磺酰氟和5,5'-二硫代双（2 - 硝基苯甲酸）的抑制，这是其他酰胺水解酶的典型特征。胞质组分中的酰胺水解酶活性对NAE18:2底物表现出饱和动力学，表观K(m)为65 μM，V(max)为83 nmol min⁻¹ mg⁻¹蛋白质。种子吸胀过程中总NAE酰胺水解酶活性增加，在开始吸水2小时后测得的活性水平最高（约为干种子中的四倍）。NAEs属于“内源性大麻素”家族，在其他类型的真核细胞中已被鉴定为有效的脂质介质。这增加了它们在植物中吸胀诱导的代谢参与种子萌发调控的可能性。
