5-lipoxygenase binds calcium

5-Lipoxygenase (5LO) catalyzes the first two steps in the biosynthesis of l eukotrienes and lipoxins and has therefore become an important target for p harmacological treatment of inflammatory disorders. Binding of calcium to 5 LO was shown using several different approaches. Human recombinant enzyme w as expressed in E. coli and purified. Association of Ca2+ to 5LO was demons trated by a calcium-induced mobility shift in gel electrophoresis, by calci um overlay, by gel filtration in the presence of calcium, and by equilibriu m dialysis. The two latter methods also showed that calcium binds reversibl y to 5LO. Equilibrium dialysis gave a K-d close to 6 mu M; the stoichiometr y of maximum calcium binding seemed to average around two Ca2+ per 5LO. Sim ilar results were obtained when 5LO was inactivated during equilibrium dial ysis, indicating that the calcium binding site(s) is (are) different from t he active site. By Triton X-114 partitioning, it was confirmed that calcium increases the hydrophobicity of 5LO.