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首页> 外文期刊>Biochemistry >Calcium is not required for 5-lipoxygenase activity at high phosphatidyl choline vesicle concentrations
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Calcium is not required for 5-lipoxygenase activity at high phosphatidyl choline vesicle concentrations

机译：在高磷脂酰胆碱囊泡浓度下，5-脂氧合酶活性不需要钙

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摘要

5-Lipoxygenase (5-LO) catalyzes the formation of 5-hydroperoxy-eicosatetraenoic acid (5 HPETE) and leukotriene A(4) (LTA(4)) from arachidonic acid. Following a rise in intracellular calcium, 5-LO translocates to a membrane where it reacts with arachidonic acid via an 18 kD protein (FLAP). In vitro studies using a vesicle system of phosphatidylcholine (PC) and purified 5-LO were conducted under varying concentrations of PC and calcium. At high PC concentrations, 5-LO partitioned onto the vesicle containing arachidonic acid, resulting in product formation in the absence of calcium. Addition of calcium increased the initial rate of the reaction with a small increase in product accumulation. Dilution experiments in the absence of calcium at high PC concentrations indicated that binding of 5-LO to the vesicles is rapidly reversible. In the presence of calcium, this binding is much more favorable than without calcium. Stimulation of 5-LO activity by dithiothreitol (DTT) was more pronounced at high PC concentrations than at low PC concentrations. The requirement for ATP for maximal activity was independent of vesicle concentration. Inhibitors that functioned in the conditions of low PC with calcium present also inhibited under high PC without calcium. In the presence of PC and calcium and without substrate, the enzyme was unstable and was rapidly and irreversibly inactivated. In high PC without calcium, the enzyme was much more stable but it was still subject to turnover-dependent inactivation. Fluorescence energy-transfer experiments confirmed the kinetic findings that 5-LO could bind to the vesicle in the absence of calcium. These results show that in the absence of calcium, 5-LO can reversibly bind to the vesicle containing arachidonic acid and produce the same amount of product by a similar mechanism as observed with low PC and calcium. Calcium likely causes a conformational change that increases the affinity of the enzyme for the vesicle, but it is not strictly required for enzymatic activity and has no effect on the function of the catalytic site. [References: 31]
机译：5-脂氧合酶（5-LO）催化从花生四烯酸形成5-氢过氧二十碳四烯酸（5 HPETE）和白三烯A（4）（LTA（4））。细胞内钙升高后，5-LO易位到膜上，并通过18 kD蛋白（FLAP）与花生四烯酸反应。在不同浓度的PC和钙浓度下，使用磷脂酰胆碱（PC）和纯化的5-LO囊泡系统进行了体外研究。在高PC浓度下，5-LO分配到含有花生四烯酸的囊泡上，导致在没有钙的情况下形成产物。钙的添加增加了反应的初始速率，而产物的积累略有增加。在高PC浓度下不含钙的稀释实验表明，5-LO与囊泡的结合是快速可逆的。在钙存在下，这种结合比没有钙时更为有利。在高PC浓度下，二硫苏糖醇（DTT）对5-LO活性的刺激比在低PC浓度下更明显。最大活性所需的ATP需求与囊泡浓度无关。在钙含量低的条件下起作用的抑制剂在钙含量高的情况下也被抑制。在PC和钙存在且没有底物的情况下，该酶不稳定，并且迅速且不可逆地失活。在不含钙的高PC中，该酶更加稳定，但仍会发生依赖于周转的失活。荧光能量转移实验证实了在没有钙的情况下5-LO可以结合到囊泡的动力学发现。这些结果表明，在没有钙的情况下，5-LO可以可逆地结合到含有花生四烯酸的囊泡上，并且通过与低PC和钙时观察到的相似机理产生相同量的产物。钙可能引起构象变化，从而增加了酶对囊泡的亲和力，但酶活性并非严格要求，并且对催化位点的功能没有影响。 [参考：31]

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