PubMed · 1921840
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G Schmolke. 1991. [Leukotrienes].. https://pubmed.ncbi.nlm.nih.gov/1921840/
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Human 5-lipoxygenase is a non-heme iron protein which is reported to be highly unstable in the presence of oxygen. The results of this investigation demonstrate that H2O2 generated during air oxidation of thiols is the main factor in non-turnover-dependent inactivation of purified recombinant human 5-lipoxygenase for the following reasons: catalase protects against oxygen-dependent inactivation of the enzyme in the presence of dithiothreitol; the active, stable enzyme can be prepared under aerobic conditions with the exclusion of dithiothreitol and contaminating metal ions; 10 microM H2O2 causes the rapid inactivation of the enzyme. The native (ferrous) enzyme is approximately seven times more sensitive to inactivation by H2O2 than the ferric enzyme, suggesting that the mechanism of inactivation involves a Fenton-type reaction of the ferrous enzyme with H2O2, resulting in the formation of an activated oxygen species. Purification of 5-lipoxygenase under aerobic conditions (no dithiothreitol) results in an increase in both the specific activity of the purified protein [up to 70 mumol 5(S)-hydroperoxy-6-trans-8, 11, 14-cis-icosatetraenoic acid (5-HPETE)/mg protein] and in the ratio of specific activity to enzyme iron content compared to enzyme purified under anaerobic conditions in the presence of dithiothreitol. The reaction of the highly active 5-lipoxygenase enzyme shows a dependence on physiological intracellular calcium concentrations, half-maximal product formation being obtained at 0.9 microM free Ca2+. The maximal enzyme activity is also dependent on EDTA and dithiothreitol and low amounts of carrier protein, as well as the known activators PtdCho and ATP. Ca2+ can be substituted by Mn2+, Ba2+ and Sr2+, although lower levels of stimulation are obtained. 5-Lipoxygenase is strongly inhibited by low concentrations (< or = 10 microM) of Zn2+ and Cu2+. The inhibition by Cu2+ is apparently irreversible, whereas that by Zn2+ is slowly reversed (t1/2 = 2 min) in the presence of excess EDTA. These observations on the mechanism of non-turnover-dependent inactivation of 5-lipoxygenase, and the optimisation of assay conditions, have facilitated the purification of large quantities of relatively stable enzyme that will be useful for further kinetic and physical studies.
Ionophore activation of the human polymorphonuclear neutrophil results in eicosanoid synthesis and the accumulation of inactive 5-lipoxygenase in a membrane compartment. We report here that inhibition of self-inactivation of 5-lipoxygenase in ionophore-treated neutrophils with the reversible inhibitor zileuton, results in the accumulation of active 5-lipoxygenase in the membrane fraction. In zileuton plus ionophore-treated cells, 77% of the specific activity of the cytosolic enzyme from resting cells was diverted to the membrane fraction compared to 22% of the activity translocated when ionophore alone was used to activate the neutrophils. Accumulation of active membrane-associated 5-lipoxygenase was inhibited and reversed by the 5-lipoxygenase translocation inhibitor MK-886. The membrane-associated 5-lipoxygenase was two times more efficient in the production of leukotriene A4 from arachidonate-derived 5-hydroperoxyeicosatetraenoic acid than the cytosolic enzyme. Unlike the cytosolic enzyme, membrane-associated 5-lipoxygenase could metabolize 12(S)- and 15(S)-hydroxyeicosatetraenoic acid to 5(S),12(S)- and 5(S),15(S)-dihydroxyeicosatetraenoic acid, respectively. The ability to metabolize hydroxy fatty acids was dependent upon 5-lipoxygenase-activating protein association, but was lost if 5-lipoxygenase was eluted from the membrane by MK-886. These studies reveal for the first time that significant quantities of active 5-lipoxygenase can be detected in the membrane fraction of activated neutrophils and show that membrane association can alter the substrate specificity of 5-lipoxygenase which is further evidence for the role of the membrane-associated enzyme in the synthesis of 5-lipoxygenase metabolites.
The biosynthesis of leukotrienes is known to occur through a series of complex processes which, in part, can be influenced by cell-cell interactions. Several studies have suggested that arachidonic acid availability is a major limiting step for leukotriene biosynthesis and that its transfer between cells can represent a significant source of this precursor. Accordingly, effect of time and source of arachidonic acid on transcellular leukotriene synthesis was studied in mixed platelet/neutrophil populations challenged with the calcium ionophore A23187. A time-dependent contribution of platelet-derived as well as neutrophil-derived arachidonate was found in the selective formation of neutrophil 5-lipoxygenase metabolites. Utilization of platelet or neutrophil arachidonate was followed by incorporation of radiolabeled arachidonic acid into platelet or neutrophil phospholipids prior to stimulation. Specific activity of liberated arachidonic acid along with numerous 5-lipoxygenase products (including LTB4, 20-hydroxy-LTB4, 5-HETE and LTC4) was determined in order to follow mass and radiolabel. A large amount of platelet-derived arachidonic acid was released in the first 1.5 min, whereas 10 min platelet-derived arachidonate was much lower in amount but significantly higher in specific activity, suggesting different precursor pools. The platelet-derived arachidonate was heavily utilized by the neutrophils at the early time points for formation of 5-HETE and delta 6-trans-LTB4 isomers, but appeared to contribute only marginally to the constitutive metabolism of neutrophil arachidonate into LTB4. Results from these experiments suggest different pools of 5-lipoxygenase in the neutrophil and indicate a time and source dependent modulation of arachidonate metabolism in mixed cell interactions.