5-lipoxygenase binds calcium.
5-Lipoxygenase (5LO) catalyzes the first two steps in the biosynthesis of leukotrienes and lipoxins and has therefore become an important target for pharmacological treatment of inflammatory disorders. Binding of calcium to 5LO was shown using several different approaches. Human recombinant enzyme was expressed in E. coli and purified. Association of Ca2+ to 5LO was demonstrated by a calcium-induced mobility shift in gel electrophoresis, by calcium overlay, by gel filtration in the presence of calcium, and by equilibrium dialysis. The two latter methods also showed that calcium binds reversibly to 5LO. Equilibrium dialysis gave a Kd close to 6 microM; the stoichiometry of maximum calcium binding seemed to average around two Ca2+ per 5LO. Similar results were obtained when 5LO was inactivated during equilibrium dialysis, indicating that the calcium binding site(s) is (are) different from the active site. By Triton X-114 partitioning, it was confirmed that calcium increases the hydrophobicity of 5LO.
Hammarberg
T
T
Department of Medical Biochemistry and Biophysics, Division of Chemistry II, Karolinska Institute, Stockholm, Sweden.
Rådmark
O
O
D001094:Arachidonate 5-Lipoxygenase / Q000737:chemistry* / Q000378:metabolism; D001345:Autoradiography; D002118:Calcium / Q000378:metabolism*; D002132:Calcium Radioisotopes; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D002850:Chromatography, Gel; D003956:Dialysis; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D011485:Protein Binding; D013025:Glycine max / Q000201:enzymology
D002132:Calcium Radioisotopes; D002135:Calcium-Binding Proteins; D001094:Arachidonate 5-Lipoxygenase; D002118:Calcium