Identification of a sphingolipid-specific phospholipase D activity associated with the generation of phytoceramide-1-phosphate in cabbage leaves.
Identification of a sphingolipid-specific phospholipase D activity associated with the generation of phytoceramide-1-phosphate in cabbage leaves.
复制标题
鉴定与甘蓝叶中植物神经酰胺-1-磷酸生成相关的鞘脂特异性磷脂酶 D 活性。
DOI：
10.1111/febs.12374
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发表时间：
2013
期刊：
影响因子：
--
通讯作者：
Minoru Nagano and Akira Tokumura
中科院分区：
文献类型：
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作者：
Tamotsu Tanaka;Takashi Kida ;Hiroyuki lmai;Jun-ichi Morishige;Sachika Uozumi;Kiyoshi Satouchi;Minoru Nagano and Akira Tokumura
用薄层层析法测定了甘蓝脂类中一种未知的脂类(脂类X)的结构和生物合成路线。Liped X是一种耐弱碱性的磷脂，可通过MALDI-TOF MS检测到与磷酸盐捕获锌络合物PHOS-TAG的加合物。以脂肪αX为原料制备脂肪酸甲酯，用气相色谱-质谱法测定了不同脂肪酸(16：0、22：0、24：0和24：1)的含量。用MALDI-TOF MS结合PHOS-TAG测定了脂肪X的脱酰基衍生物为4-羟基鞘氨醇(脱氢磷脂)-1-磷酸。根据这些结果，确定脂质X是含有α-羟基脂肪酸的植物神经酰胺-1-磷酸(PC1P)。当甘蓝匀浆培养时，PC1P的形成伴随着糖肌醇磷酰胺(GIPC)的减少。用甘蓝匀浆的膜部分处理纯化的甘蓝GIPC，证实了GIPC中PC1P的形成。利用部分纯化的酶组分，我们发现该酶特异性地降解GIPC，而不是甘油磷脂和鞘磷脂。拟南芥也具有这种酶的活性。根据这些结果，我们得出结论，在十字花科植物中，一种以前未被描述的磷脂酶和D活性特异性地降解GIPC产生PC1P。
The structure and biosynthetic route for an unidentified lipid (lipid X) detected by TLC of cabbage (Brassica oleracea) lipids was determined. Lipid X is a phospholipid that is resistant to mild alkali and detectable by MALDI‐TOF MS as an adduct with Phos‐tag, a phosphate‐capture zinc complex. Various α‐hydroxy fatty acids (16:0, 22:0, 24:0 and 24:1) were detected by GC‐MS of fatty acid methyl esters prepared from lipid X. The deacyl derivative of lipid X was determined to be 4‐hydroxysphingenine (dehydrophytosphingosine)‐1‐phosphate by MALDI‐TOF MS with Phos‐tag. From these results, lipid X was determined to be phytoceramide‐1‐phosphate (PC1P) with an α‐hydroxy fatty acid. When cabbage homogenates were incubated, PC1P was formed, with a concomitant decrease in the amount of glycosylinositol phosphoceramide (GIPC). The formation of PC1P from GIPC was confirmed by treatment of purified cabbage GIPC with a membrane fraction of cabbage homogenates. Using a partially purified enzyme fraction, we found that the enzyme hydrolyzes GIPC specifically, but not glycerophospholipids and sphingomyelin.Arabidopsis thalianaalso had this enzyme activity. From these results, we conclude that a previously uncharacterized phospholipase D activity that specifically hydrolyzes GIPC produces PC1P in brassicaceous plants.