Murray J J, Brash A R
Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee 37232.
Arch Biochem Biophys. 1988 Sep;265(2):514-23. doi: 10.1016/0003-9861(88)90156-7.
The rabbit reticulocyte lipoxygenase is known to display an unusual facility for oxygenation of esterified polyunsaturated fatty acids, yet the precise structures of the products are not known. With free arachidonate as substrate the enzyme is known to catalyze 15S and 12S oxygenations, and demonstration of a facility for catalysis of these reactions on phospholipids would extend the potential scope of lipoxygenase reactions in cells. We elected to study in detail the reaction of the enzyme with a natural phospholipid, palmitoyl/arachidonoyl-phosphatidylcholine. We determined the nature of the products by initial isolation by RP-HPLC, followed by transesterification and identification of the oxygenated products by HPLC, uv, GC-MS, and steric analysis of hydroxyl configuration by HPLC. The major product was identified as a phosphatidylcholine in which the arachidonate component was converted to the 15(S)-hydroperoxy-eicosatetraenoate. A second oxygenated phospholipid was produced in smaller quantities (2-5% of the latter product) and identified as the 12(S)-oxygenated analog. These products were also identified after reaction of the reticulocyte lipoxygenase with human red cell membranes which were radiolabeled by preincubation with [3H]arachidonic acid. The finding of 12S oxygenation represents the first evidence that a lipoxygenase can control a reaction centered on the 10-carbon of an arachidonoyl phospholipid. This is an important precedent, because hydrogen abstraction from carbon-10 is a critical step in the lipoxygenase-catalyzed synthesis of 8- and 12-hydroperoxy-eicosatetraenoates (HPETEs) and for the conversion of 5- and 15-HPETEs to leukotrienes.
已知兔网织红细胞脂氧合酶对酯化多不饱和脂肪酸的氧化具有非凡的能力，但产物的确切结构尚不清楚。以游离花生四烯酸为底物时，已知该酶可催化15S和12S氧化反应，若能证明其对磷脂上这些反应具有催化能力，将扩大细胞中脂氧合酶反应的潜在范围。我们选择详细研究该酶与天然磷脂棕榈酰/花生四烯酰磷脂酰胆碱的反应。我们首先通过反相高效液相色谱（RP-HPLC）初步分离产物，然后进行酯交换反应，再通过高效液相色谱、紫外光谱、气相色谱-质谱联用以及利用高效液相色谱对羟基构型进行空间分析来鉴定氧化产物的性质。主要产物被鉴定为一种磷脂酰胆碱，其中花生四烯酸成分转化为15(S)-氢过氧化二十碳四烯酸。还产生了少量的第二种氧化磷脂（占后一种产物的2 - 5%），并鉴定为12(S)-氧化类似物。在用[3H]花生四烯酸预孵育进行放射性标记的人红细胞膜与网织红细胞脂氧合酶反应后，也鉴定出了这些产物。12S氧化的发现代表了首个证据，表明脂氧合酶可以控制以花生四烯酰磷脂的10位碳原子为中心的反应。这是一个重要的先例，因为从碳-10上夺取氢是脂氧合酶催化合成8-和12-氢过氧化二十碳四烯酸（HPETEs）以及将5-和15-HPETEs转化为白三烯的关键步骤。
