Development of a fluorescence-based enzyme assay of human 5-lipoxygenase

Pufahl, Robert A.; Kasten, Tom; Hills, Rob; Gierse, James K.; Reitz, Beverly A.; Weinberg, Robin A.; Masferrer, Jaime L.

doi:10.1016/j.ab.2007.02.009

Cited by 54 publications

(39 citation statements)

References 33 publications

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“…FITC binding leads to a decrease in enzyme activity, thus making it inappropriate for activity detection. In contrast to FITC, which detects LOX protein, use of H 2 DCFDA provides advantage since it detects LOX activity [16,17]. In recent past, H 2 DCFDA has been used to investigate membrane lipid peroxidation in roots of Arabidopsis [18].…”

Section: Introductionmentioning

confidence: 97%

“…Intracellular levels of hydroperoxides have also been determined in human cells using H 2 DCFDA, followed by flow cytometric analysis [19]. Recently, a fluorescence-based assay system, using H 2 DCFDA has been developed for estimation of LOX activity [17]. This method satisfies a number of parameters to be convincingly used as a detection agent for LOX activity.…”

Section: Introductionmentioning

confidence: 99%

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Localization of lipoxygenase activity on the oil bodies and in protoplasts using a novel fluorescence imaging method

Yadav

Bhatla

2011

Plant Physiology and Biochemistry

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Section: Introductionmentioning

confidence: 97%

Section: Introductionmentioning

confidence: 99%

Localization of lipoxygenase activity on the oil bodies and in protoplasts using a novel fluorescence imaging method

Yadav

Bhatla

2011

Plant Physiology and Biochemistry

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“…The enzyme assay is based on the oxidation of the substrate H 2 DCFDA to the highly fluorescent 2Ј,7Ј-dichlorofluorescein product as described previously (Pufahl et al, 2007). The enzyme assay (40 l) contained 50 mM Tris, pH 7.5, 2 mM EDTA, 2 mM CaCl 2 , 3 M AA, 10 M ATP, 10 M H 2 DCFDA, and recombinant enzyme (8 g of lysate).…”

Section: Methodsmentioning

confidence: 99%

“…Initial rate measurements of 5-LOX activity were determined by UV absorption of conjugated diene products (5-hydroperoxyeicosatetraenoic acid and 5-HETE) formation at 238 nm as described previously (Pufahl et al, 2007). Assays (1 ml) contained 50 mM potassium phosphate, pH 7.6, 0.1 mM EDTA, 0.3 mM CaCl 2 , 20 M AA, 100 M ATP, inhibitor, and human 5-LOX enzyme (75 g of lysate).…”

Section: Methodsmentioning

confidence: 99%

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Pharmacology of PF-4191834, a Novel, Selective Non-Redox 5-Lipoxygenase Inhibitor Effective in Inflammation and Pain

Masferrer

Zweifel²,

Hardy³

et al. 2010

J Pharmacol Exp Ther

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5-Lipoxygenase (LOX) is an important arachidonic acidmetabolizing enzyme producing leukotrienes and other proinflammatory lipid mediators with potent pathophysiological functions in asthma and other inflammatory diseases. 4-(3-(4-(1-Methyl-1H-pyrazol-5-yl)phenylthio)phenyl)-tetrahydro-2H-pyran-4-carboxamide (PF-4191834) is a novel, selective non-redox 5-lipoxygenase inhibitor effective in inflammation and pain. In vitro and in vivo assays were developed for the evaluation of a novel 5-LOX inhibitor using conditions of maximal enzyme activity. PF-4191834 exhibits good potency in enzyme-and cell-based assays, as well as in a rat model of acute inflammation. Enzyme assay results indicate that PF-4191834 is a potent 5-LOX inhibitor, with an IC 50 ϭ 229 Ϯ 20 nM. Furthermore, it demonstrated ϳ300-fold selectivity for 5-LOX over 12-LOX and 15-LOX and shows no activity toward the cyclooxygenase enzymes. In addition, PF-4191834 inhibits 5-LOX in human blood cells, with an IC 80 ϭ 370 Ϯ 20 nM. This inhibitory concentration correlates well with plasma exposures needed for in vivo efficacy in inflammation in models of inflammatory pain. The combination of potency in cells and in vivo, together with a sustained in vivo effect, provides PF-4191834 with an overall pharmacodynamic improvement consistent with once a day dosing.The 5-lipoxygenase (LOX) pathway is thought to play an important role in the pathophysiology of asthma and other inflammatory diseases by controlling the production of several key inflammatory mediators (Harris et al., 1995;Rastogi and McHowat, 2006;Peters-Golden and Henderson, 2007;Rubin and Mollison, 2007). 5-LOX is required for the production of leukotrienes C 4 , D 4 , and E 4 (collectively known as the cysteinyl leukotrienes; cys-LTs), which are potent bronchoconstrictors and proinflammatory mediators. Cys-LTs are generated after5-LOX metabolism of arachidonic acid (AA) to form leukotriene (LT)A 4 . Another enzyme, LTC 4 synthase, present in several cells, including eosinophils, basophils, and mast cells, conjugates LTA 4 to glutathione to yield LTC 4 . LTC 4 is further metabolized into LTD 4 and LTE 4 (Samuelsson, 1987). The 5-LOX enzyme is also involved in the production of LTB 4 , a primary attractant and activator for leukocytes. This lipid is primarily synthesized in neutrophils and macrophages where the enzyme LTA 4 hydrolase converts LTA 4 to the potent chemoattractant LTB 4 (Samuelsson, 1987). 5-LOX activity also results in the production of bioactive metabolites 5-hydroxyeicosatetraenoic acid (HETE) and 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxoETE) (Miller et al., 2000;Powell and Rokach, 2005). 5-oxoETE has been shown to induce tissue eosinophilia; thus, it may play a role in asthma and other diseases (Stamatiou et al., 1998;Guilbert et al., 1999;Muro et al., 2003).The clinical importance of the leukotriene pathway in inflammatory airway disease is demonstrated by the efficacy of various agents in the treatment of asthma and allergic rhinitis. Cysteinyl leukotriene receptor 1 anta...

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Human 5‐Lipoxygenase

Newcomer

Gilbert

Funk

2012

Encyclopedia of Inorganic and Bioinorganic Chemistry

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The enzyme 5‐lipoxygenase initiates the biosynthesis of leukotrienes, lipid mediators of the inflammatory response. The enzyme is also required for the synthesis of lipoxins, arachidonic‐acid‐derived compounds that promote resolution of inflammation. Lipoxygenases are non‐heme‐iron enzymes that catalyze the peroxidation of polyunsaturated fatty acids. Although all arachidonate‐metabolizing lipoxygenases recognize the same 20‐carbon polyunsaturated fatty acid, each generates a unique product. We describe here the structure of 5‐lipoxygenase, which is distinct among lipoxygenases with respect to the reactions it catalyzes and its regulation. Multiple regulatory mechanisms that combine to ensure a measured generation of its products, precursors of potent signaling compounds, are described. These mechanisms include an atypical (with respect to the lipoxygenase superfamily) protein lability, Ca 2+ ‐induced translocation to the nuclear membrane, and serine phosphorylation.

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Development of a fluorescence-based enzyme assay of human 5-lipoxygenase

Cited by 54 publications

References 33 publications

Localization of lipoxygenase activity on the oil bodies and in protoplasts using a novel fluorescence imaging method

Localization of lipoxygenase activity on the oil bodies and in protoplasts using a novel fluorescence imaging method

Pharmacology of PF-4191834, a Novel, Selective Non-Redox 5-Lipoxygenase Inhibitor Effective in Inflammation and Pain

Human 5‐Lipoxygenase

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