Malaviya R, Malaviya R, Jakschik B A
Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110.
J Biol Chem. 1993 Mar 5;268(7):4939-44.
The 5-lipoxygenase catalyzes the first two steps in the metabolism of arachidonic acid to leukotrienes. It has been shown that the calcium influx into leukocytes following stimulation by A23187 activates the enzyme and causes its translocation to the membrane. Leukotrienes are formed, and then the enzyme seems to be irreversibly inactivated. In the present investigation we have compared the effect of receptor-mediated mast cell activation (IgE/antigen) and A23187 on 5-lipoxygenase activity and translocation to the membrane. In contrast to the ionophore, IgE/antigen, which initiated the formation of similar amounts of leukotrienes as A23187, caused minor inactivation of the enzyme. Immunoblot analysis demonstrated that after antigen the membrane association of the 5-lipoxygenase was reversible, while with A23187 the translocation continued during the time of observation (60 min). Addition of a calcium chelator after ionophore challenge, prevented further inactivation of the enzyme and reversed its membrane binding. The data suggest that the continuous influx of calcium with A23187 is responsible for the extensive inactivation of the 5-lipoxygenase. In contrast, during receptor-mediated stimulation the transient increase in intracellular calcium seems to conserve the enzyme activity.
5-脂氧合酶催化花生四烯酸代谢生成白三烯的前两个步骤。研究表明，A23187刺激白细胞后，钙离子内流会激活该酶并使其转位至细胞膜。白三烯生成后，该酶似乎会不可逆地失活。在本研究中，我们比较了受体介导的肥大细胞活化（IgE/抗原）和A23187对5-脂氧合酶活性及转位至细胞膜的影响。与离子载体不同，IgE/抗原引发的白三烯生成量与A23187相似，但导致该酶的失活程度较小。免疫印迹分析表明，抗原刺激后，5-脂氧合酶与细胞膜的结合是可逆的，而使用A23187时，在观察期间（60分钟）转位仍在继续。离子载体刺激后添加钙螯合剂可防止该酶进一步失活并逆转其与细胞膜的结合。数据表明，A23187引起的钙离子持续内流是5-脂氧合酶广泛失活的原因。相比之下，在受体介导的刺激过程中，细胞内钙离子的短暂增加似乎能保持该酶的活性。
