Chapman K D, Moore T S
Botany Department, Louisiana State University, Baton Rouge 70803-1705.
Arch Biochem Biophys. 1993 Feb 15;301(1):21-33. doi: 10.1006/abbi.1993.1110.
We have identified a microsomal phospholipid as N-acylphosphatidylethanolamine (NAPE) that was radiolabeled following incubation of 1-day-old cotyledons of cotton seedlings with [14C]ethanolamine. Radiolabeled NAPE comigrated with commercially available L-alpha-dipalmitoyl phosphatidyl(N-palmitoyl)ethanolamine (std-NAPE) in one- and two-dimensional TLC. This NAPE comprised 7.2 +/- 1.0% (by weight) of microsomal phospholipids when hot isopropanol was used to inactivate endogenous phospholipases prior to extraction of lipids. In vitro degradation of putative cottonseed radiolabeled NAPE by Streptomyces chromofuscus phospholipase D resulted in production of a ninhydrin-reactive, radiolabeled lipid which comigrated with N-acylethanolamine (NAE) that was produced from a similar enzymatic cleavage of std-NAPE. Transmethylation of cottonseed radiolabeled NAE yielded radiolabeled ethanolamine and fatty acid methyl esters, nearly all of which were saturated (myristate, palmitate, and stearate together were 92% of the acyl components of cottonseed NAE). Positional analysis and relative abundance of the O-acyl groups of cottonseed microsomal NAPE were determined following a double enzymatic cleavage with Trimeresurus flavoviridis venom (phospholipase A2 activity) and S. chromofuscus phospholipase D. We substantiated our identification of cottonseed NAPE by 1H NMR spectroscopy and by mass spectrometry (fast-atom-bombardment ionization and tandem MS, FAB-MS/MS). Radiolabeled NAPE was synthesized in vivo in varying amounts from [14C]ethanolamine applied to maturing seeds of cotton and soybean, cotyledons of dark-grown cotton and soybean seedlings, cotyledons of light-grown okra, cotton and soybean seedlings, endosperm tissue of castor bean, and suspension cell cultures of rice. In pulse-chase radiolabeling experiments in cotyledons of 1-day-old cotton seedlings, radiolabeled NAPE increased and radiolabeled phosphatidylethanolamine (PE) decreased over a 12-h chase period (in the dark or light), suggesting that NAPE was synthesized from PE in vivo. In vitro, the synthesis of NAPE from PE (radiolabeled in vivo) proceeded in a linear fashion in microsomes of cotton cotyledons with or without 1 mM EGTA and with or without 1 mM CaCl2 for 90 min. NAPE was synthesized in vitro from PE synthesized by the exchange pathway (microsomes preincubated with [14C]++e+thanolamine) and from PE synthesized by the nucleotide pathway (microsomes preincubated with [14C]CDPethanolamine). Collectively, our data indicate that (a) NAPE is a widespread, natural phospholipid component of plants, (b) NAPE is synthesized in vivo under normal physiological growth conditions in cotyledons of cotton seedlings, (c) NAPE is localized and synthesized in cottonseed microsomes, and (d) NAPE is likely synthesized by a direct acylation of PE.
我们已鉴定出一种微粒体磷脂为N-酰基磷脂酰乙醇胺（NAPE），在用[14C]乙醇胺孵育棉花幼苗1日龄子叶后，该磷脂被放射性标记。在一维和二维薄层层析中，放射性标记的NAPE与市售的L-α-二棕榈酰磷脂酰（N-棕榈酰）乙醇胺（标准NAPE）迁移率相同。当在脂质提取前用热异丙醇使内源性磷脂酶失活时，这种NAPE占微粒体磷脂的7.2±1.0%（重量）。深褐链霉菌磷脂酶D对假定的棉花种子放射性标记NAPE进行体外降解，产生了一种与茚三酮反应的放射性标记脂质，其迁移率与由标准NAPE的类似酶促裂解产生的N-酰基乙醇胺（NAE）相同。棉花种子放射性标记NAE的甲基化产生了放射性标记的乙醇胺和脂肪酸甲酯，其中几乎所有都是饱和的（肉豆蔻酸、棕榈酸和硬脂酸一起占棉花种子NAE酰基成分的92%）。在用竹叶青蛇毒（磷脂酶A2活性）和深褐链霉菌磷脂酶D进行双重酶促裂解后，测定了棉花种子微粒体NAPE的O-酰基的位置分析和相对丰度。我们通过1H核磁共振光谱和质谱（快原子轰击电离和串联质谱，FAB-MS/MS）证实了我们对棉花种子NAPE的鉴定。放射性标记的NAPE在体内由施用于棉花和大豆成熟种子、黑暗生长的棉花和大豆幼苗子叶、光照生长的秋葵、棉花和大豆幼苗、蓖麻子胚乳组织以及水稻悬浮细胞培养物中的[14C]乙醇胺以不同量合成。在1日龄棉花幼苗子叶的脉冲追踪放射性标记实验中，在12小时的追踪期内（在黑暗或光照下），放射性标记的NAPE增加而放射性标记的磷脂酰乙醇胺（PE）减少，这表明NAPE在体内由PE合成。在体外，无论有无1 mM乙二醇双乙醚四乙酸（EGTA）和有无1 mM氯化钙（CaCl2），在棉花子叶微粒体中，由体内放射性标记的PE合成NAPE以线性方式进行90分钟。NAPE在体外由通过交换途径合成的PE（微粒体预先与[14C]乙醇胺预孵育）和由核苷酸途径合成的PE（微粒体预先与[14C]CDP乙醇胺预孵育）合成。总体而言，我们的数据表明：（a）NAPE是植物中广泛存在的天然磷脂成分；（b）NAPE在棉花幼苗子叶的正常生理生长条件下在体内合成；（c）NAPE定位于棉花种子微粒体并在其中合成；（d）NAPE可能通过PE的直接酰化合成。
