Complex sphingolipid synthesis in plants: characterization of inositolphosphorylceramide synthase activity in bean microsomes
Complex sphingolipid synthesis in plants: characterization of inositolphosphorylceramide synthase activity in bean microsomes
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DOI：
10.1016/s0003-9861(03)00339-4
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发表时间：
2003-09-15
影响因子：
3.9
通讯作者：
Lynch, DV
中科院分区：
文献类型：
--
作者：
Bromley, PE;Li, YNO;Lynch, DV
存在于植物中的复杂糖磷鞘脂由神经酰胺、肌醇磷酸和多种极性寡糖取代基组成。肌醇磷酸神经酰胺（IPC）合酶（磷脂酰肌醇：神经酰胺肌醇磷酸转移酶）的活性，提出催化这些复杂的鞘脂形成的初始承诺步骤的酶，其特征在于在蜡豆下胚轴微粒体。通过监测荧光NBD-C-6神经酰胺或来自放射性标记的磷脂酰肌醇（PI）的[H-3]肌醇磷酸掺入TLC鉴定的产物中来测定酶活性。发现IPC合成酶利用含非羟基脂肪酸的神经酰胺、含羟基脂肪酸的神经酰胺和NBD-C-6神经酰胺作为底物。在PI浓度超过600 μ M时观察到最大产物形成（在约200 μ M时具有半最大活性）。内源性PI和神经酰胺似乎作为底物。真菌IPC合成酶的两种有效抑制剂Aureobasidin A和rustmicin抑制豆微粒体中的酶活性，IC 50值分别为0.4-0-8和16-20 nM。IPC合酶活性出现最密切相关的高尔基体的基础上使用选定的标记酶的结果。在多种植物组织中检测到酶活性。这份报告，第一次表征IPC合成酶在植物组织中，证明了植物酶和酵母菌对应物之间的相似性，并提供了深入了解植物glycophosphingolipid生物学。(C)2003年爱思唯尔公司All rights reserved.
Complex glycophosphosphingolipids present in plants are composed of ceramide, inositolphosphate, and diverse polar oligosaccharide substituents. The activity of inositolphosphorylceramide (IPC) synthase (phosphatidylinositol:ceramide inositolphosphate transferase), the enzyme proposed to catalyze the initial committed step in the formation of these complex sphingolipids, was characterized in wax bean hypocotyl microsomes. Enzyme activity was assayed by monitoring the incorporation of fluorescent NBD-C-6 ceramide or [H-3]inositolphosphate from radiolabeled phosphatidylinositol (PI) into product identified by TLC. IPC synthase was found to utilize nonhydroxy fatty acid-containing ceramide, hydroxy fatty acid-containing ceramide, and NBD-C-6 ceramide as substrate. Maximum product formation was observed at PI concentrations in excess of 600 muM (with half-maximum activity at approximately 200 muM). Both endogenous PI and ceramide appeared to serve as substrates. Aureobasidin A and rustmicin, two potent inhibitors of fungal IPC synthase, inhibited enzyme activity in bean microsomes with values for IC50 Of 0.4-0-8 and 16-20 nM, respectively. IPC synthase activity appeared most closely associated with the Golgi based on results using selected marker enzymes. Enzyme activity was detected in a variety of plant tissues. This report, the first to characterize IPC synthase in plant tissues, demonstrates the similarities between the plant enzyme and its yeast counterpart, and provides insight into plant glycophosphosphingolipid biology. (C) 2003 Elsevier Inc. All rights reserved.