Interactions of 5-lipoxygenase with membranes: studies on the association of soluble enzyme with membranes and alterations in enzyme activity

Abstract

Treatment of rat basophilic leukemia cells (RBL-1) with the calcium ionophore A23187 resulted in activation of 5-lipoxygenase, as indicated by an induction of leukotriene release [Orning. L., Hammarstrom, S., and Samuelsson, B. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 2017]. The enzyme activation was accompanied by a time-dependent association of 5-lipoxygenase to the particular fraction. When cells were lysed in the presence of 0.05-10 .mu.M CaCl2, the soluble 5-lipoxygenase became associated with the particulate fraction. This was demonstrated by a decrease in immunoreactivities and enzymatic activities in the soluble fraction and a parallel increase in particulate-associated immunoreactivities. The particulate-bound enzyme was not active. Ca2+ induced the membrane association of 5-lipoxygenase when added into the incubation mixtures containing the membrane fraction with either the cytosolic fraction or the purified enzyme. 5-Lipoxygenase also bound to the microsomal-enriched fraction in the presence of Ca2+. Maximal membrane binding was obtained after a 1-min incubation at 4.degree. C. When a fixed amount of isolated membranes (0.2 mg of protein) and increasing cytosolic protein (0.5-4 mg) were used, a linear increase in enzyme binding was observed. The binding became saturated at 3 mg of cytosolic protein/mg of membrane protein. 5-Lipoxygenase binding to the membrane fraction was unaffected by pretreatment of the membranes with trypsin but was inhibited by treating with phospholipase A2, suggesting that phospholipids are involved. The membrane-bound enzyme was not extracted by treatment with a chelator (10 mM EDTA) or high salt (2 M NaCl) but was dissociated from membranes with detergents (0.5% sodium dodecyl sulfate, 10 mM Chaps, 0.5% digitonin, 0.5% Triton X-100, or 1% Brij 35). The membrane fraction when added to the cytosol, followed by subsequent addition of Ca2+ and substrate, stimulated 5-lipoxygenase activity. There was an increase in both the extent of product formation and the pseudo-steady-state velocity. The maximal stimulation was produced by 0.2 mg/mL membranes. In summary, these data suggest that Ca2+ induces a shift of 5-lipoxygenase from a soluble to a membrane-associated form which is accompanied by activation of the enzyme. Membrane phospholipids may be the elements in the membranes that bind to 5-lipoxygenase.