O'Connor Butler E Susan, Mazerik Jessica N, Cruff Jason P, Sherwani Shariq I, Weis Barbara K, Marsh Clay B, Raghavamenon Achuthan C, Uppu Rao M, Schmid Harald H O, Parinandi Narasimham L
Division of Pulmonary, Allergy, Critical Care, and Sleep Medicine, Department of Internal Medicine, The Ohio State University College of Medicine, Columbus, OH, USA.
Methods Mol Biol. 2010;610:387-401. doi: 10.1007/978-1-60327-029-8_23.
The importance of understanding the mechanisms of modulation of cellular signaling cascades by the peroxidized membrane phospholipids (PLs) is well recognized. The enzyme-catalyzed peroxidation of PLs, as opposed to their oxidation by air and metal catalysis, is well controlled and rapid and yields well-defined PL peroxides which are highly desirable for biological studies. Therefore, here, we chose bovine liver phosphatidylinositol (PI), a crucial membrane PL which acts as the substrate for phospholipase C in cellular signal transduction, as a model membrane PL. We successfully generated the PI peroxides with soybean type-I lipoxygenase (LOX) in the presence of deoxycholate, which facilitates the LOX-mediated peroxidation of the polyunsaturated fatty acids esterified to the PL. The LOX-peroxidized PI, after enzymatic catalysis, was separated from the unoxidized PI in the reaction mixture by normal-phase, high-performance liquid chromatography (HPLC). The extent of LOX-mediated peroxidation of PI following HPLC purification was established by the analysis of lipid phosphorus, conjugated dienes by UV spectrophotometry, peroxides, and loss of fatty acids by gas chromatography. This study established the optimal conditions yielding approximately 46% of peroxidized PI from 300 microg of neat bovine liver PI that was peroxidized by soybean type-I LOX (50 microg) for 30 min in borate buffer (0.2 M, pH 9.0) containing 10 mM deoxycholate.
理解过氧化膜磷脂（PLs）对细胞信号级联反应的调节机制的重要性已得到广泛认可。与通过空气和金属催化氧化PLs不同，酶催化的PLs过氧化反应可控且迅速，能产生定义明确的PL过氧化物，这对于生物学研究非常理想。因此，在此我们选择牛肝磷脂酰肌醇（PI），一种关键的膜PL，它在细胞信号转导中作为磷脂酶C的底物，作为模型膜PL。我们在脱氧胆酸盐存在的情况下，用大豆I型脂氧合酶（LOX）成功生成了PI过氧化物，脱氧胆酸盐可促进LOX介导的与PL酯化的多不饱和脂肪酸的过氧化反应。酶催化后，通过正相高效液相色谱（HPLC）将反应混合物中LOX过氧化的PI与未氧化的PI分离。通过分析脂质磷、用紫外分光光度法测定共轭二烯、过氧化物以及用气相色谱法测定脂肪酸损失，确定了HPLC纯化后LOX介导的PI过氧化程度。本研究确定了最佳条件，即300微克纯牛肝PI在含有10 mM脱氧胆酸盐的硼酸盐缓冲液（0.2 M，pH 9.0）中，被大豆I型LOX（50微克）过氧化30分钟，可产生约46%的过氧化PI。
