Sensitivity of immunoaffinity-purified porcine 5-lipoxygenase to inhibitors and activating lipid hydroperoxides

Abstract

The requirement for hydroperoxide activation and the effect of inhibitors from different structural classes on 5-lipoxygenase activity were determined on the immunoaffinity-purified enzyme from porcine leukocytes. The 5-lipoxygenase activity was measured using a continuous spectrophotometric assay monitoring the increase in conjugated diene formation (A235) upon incubation of the enzyme with arachidonic acid. Under standard assay conditions, the reaction progress curves showed little or no lag phase, with a rapid first-order decay in enzyme activity (T1/2 = 0.7 to 1.1 min). Both the initial rate of the reaction and total product formation were stimulated by the addition of ATP, Ca2+ and phosphatidylcholine (PC). PC (24 micrograms/ml) was also found to increase the recovery of radiolabeled arachidonic acid from the assay mixture and thus part of the stimulation may be due to an increase in substrate availability and reduction of surface adsorption effects. The requirement of hydroperoxides for the initiation of the reaction was shown by the induction of 0.1 to 1-min lag phases using NaBH4 or glutathione peroxidase and by the reduction in lag times by 5-hydroperoxyeicosatetraenoic acid (5-HPETE) and 13-hydroperoxyoctadecadienoic acid (13-HPOD). The following compounds were evaluated as inhibitors of the 5-lipoxygenase reaction and caused a 50% decrease in product accumulation (IC50) at the indicated concentrations: quercetin, L-651,896, L-656,224, MTPPH and L-651,392 (0.3-0.5 microM); diphenyldisulfide (2-5 microM); phenidone (5-10 microM); AA861 (4-10 microM) and BW755C (4-15 microM). In addition, the presence of inhibitors extended the initial lag phase of the reaction and increased the dependence of the initiation of the reaction on exogenous lipid hydroperoxides. The inhibition by phenidone was accompanied by a 2-fold increase in the rate of enzyme inactivation, whereas other compounds such as AA861 and L-656,224 did not show this effect. The results indicate that the presence of inhibitors can modify the kinetics of 5-lipoxygenase at the levels of the initiation of the reaction and the rate of enzyme inactivation, with variations depending on the structural class of the inhibitor and the concentration of lipid hydroperoxides.