Abstract
Analysis of purified soybean and rabbit reticulocyte 15-lipoxygenase (15-LOX) and PA317 cells transfected with human 15-LOX revealed a rapid rate of linoleate-dependent nitric oxide (.NO) uptake that coincided with reversible inhibition of product ((13S)-hydroperoxyoctadecadienoic acid, or (13S)-HPODE) formation. No reaction of .NO (up to 2 microM) with either native (Ered) or ferric LOXs (0.2 microM) metal centers to form nitrosyl complexes occurred at these .NO concentrations. During HPODE-dependent activation of 15-LOX, there was consumption of 2 mol of .NO/mol of 15-LOX. Stopped flow fluorescence spectroscopy showed that.NO (2.2 microM) did not alter the rate or extent of (13S)-HPODE-induced tryptophan fluorescence quenching associated with 15-LOX activation. Additionally, .NO does not inhibit the anaerobic peroxidase activity of 15-LOX, inferring that the inhibitory actions of .NO are due to reaction with the enzyme-bound lipid peroxyl radical, rather than impairment of (13S)-HPODE-dependent enzyme activation. From this, a mechanism of 15-LOX inhibition by .NO is proposed whereby reaction of .NO with EredLOO. generates Ered and LOONO, which hydrolyzes to (13S)-HPODE and nitrite (NO2-). Reactivation of Ered, considerably slower than dioxygenase activity, is then required to complete the catalytic cycle and leads to a net inhibition of rates of (13S)-HPODE formation. This reaction of .NO with 15-LOX inhibited. NO-dependent activation of soluble guanylate cyclase and consequent cGMP production. Since accelerated .NO production, enhanced 15-LOX gene expression, and 15-LOX product formation occurs in diverse inflammatory conditions, these observations indicate that reactions of .NO with lipoxygenase peroxyl radical intermediates will result in modulation of both .NO bioavailability and rates of production of lipid signaling mediators.
Highlights
Heme iron-containing enzymes that oxidize the unsaturated fatty acids arachidonate and linoleate to bioactive hydroperoxides and other metabolites (Scheme 1)
Generates precursors for leukotrienes, products involved in inflammation and allergic responses [1]. 12-Lipoxygenases, present in vascular endothelium, smooth muscle cells, platelets, and leukocytes [2, 3], contribute to vascular cell hypertrophy, proliferation, and hypertensive actions, while 15-LOX is involved in cell development and differentiation, in reticulocytes where 15-LOX oxidation of mitochondrial phospholipids is a trigger for their degradation [2, 4, 5]
Soluble guanylate cyclase was not significantly inhibited by either native or oxidized arachidonate. This affirmed that 1⁄7NO reaction with and consumption by enzyme-bound peroxyl radical intermediates during catalytic cycling of 15-LOX turnover was responsible for inhibition of guanylate cyclase, rather than direct guanylate cyclase inactivation by oxidized lipid products that are formed during
Summary
Materials—Rabbit reticulocyte 15-LOX was purified to electrophoretic homogeneity from the lysate of a reticulocyte-rich blood cell suspension by fractionated ammonium sulfate precipitation and two consecutive steps of fast liquid protein chromatography [43]. 15-LOX assay was performed at 20 °C or 37 °C for the soybean and rabbit enzymes, respectively, with stirring. M) was added to sample buffer containing linoleate without enzyme. Once the electrode response had stabilized, enzyme was added and rates of 1⁄7NO consumption recorded. 1–2 ϫ 106 cells were added to 1 ml of PBS in the chamber of the 1⁄7NO electrode, at 37 °C with stirring. Nitric oxide (1.9 M) was added and consumption rates monitored with or without addition of 0.5 mM linoleate. Reaction mixtures contained 92 ng of soluble guanylate cyclase, 3 mM MgCl2, 1 mM cGMP, 0.3 mM [␣-32P]GTP (ϳ3 ϫ 105 cpm) in 0.1 ml of 50 mM triethenolamine/HCl buffer, pH 7.4. 15-LOX was added at the same time as DEA-NONOate. Results were corrected for enzyme-deficient blanks and recovery of cGMP
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