RESUMO
Human 5-lipoxygenase (5-LO-WT) initiates the leukotriene (LT) biosynthesis. LTs play an important role in diseases like asthma, atherosclerosis and in many types of cancer. In this study, we investigated the 5-LO isoforms 5-LO∆13, 5-LO∆4 and 5-LOp12, lacking the exons 13, 4 or a part of exon 12, respectively. We were able to detect the mRNA of the isoforms 5-LO∆13 and 5-LOp12 in B and T cell lines as well as in primary B and T cells and monocytes. Furthermore, we found that expression of 5-LO and particularly of the 5-LO∆13 and 5-LOp12 isoforms is increased in monocytes from patients with rheumatoid arthritis and sepsis. Confocal microscopy of HEK293T cells stably transfected with tagged 5-LO-WT and/or the isoforms revealed that 5-LO-WT is localized in the nucleus whereas all isoforms are located in the cytosol. Additionally, all isoforms are catalytically inactive and do not seem to influence the specific activity of 5-LO-WT. S271A mutation in 5-LO-WT and treatment of the cells with sorbitol or KN-93/SB203580 changes the localization of the WT enzyme to the cytosol. Despite colocalization with the S271A mutant, the isoforms did not affect LT biosynthesis. Analysis of the phosphorylation pattern of 5-LO-WT and all the isoforms revealed that 5-LOp12 and 5-LO∆13 are highly phosphorylated at Ser271 and 5-LOp12 at Ser523. Furthermore, coexpression of the isoforms inhibited or stimulated 5-LO-WT expression in transiently and stably transfected HEK293T cells suggesting that the isoforms have other functions than canonical LT biosynthesis.
Assuntos
Araquidonato 5-Lipoxigenase/metabolismo , Núcleo Celular/ultraestrutura , Citosol/ultraestrutura , Isoformas de Proteínas/metabolismo , Araquidonato 5-Lipoxigenase/química , Araquidonato 5-Lipoxigenase/isolamento & purificação , Núcleo Celular/metabolismo , Citosol/metabolismo , Regulação Enzimológica da Expressão Gênica , Células HEK293 , Humanos , Leucotrienos/biossíntese , Neutrófilos/metabolismo , Neutrófilos/ultraestrutura , Fosforilação , Isoformas de Proteínas/química , Isoformas de Proteínas/isolamento & purificaçãoRESUMO
Leukotrienes (LTs) are inflammatory mediators that play a pivotal role in many diseases like asthma bronchiale, atherosclerosis and in various types of cancer. The key enzyme for generation of LTs is the 5-lipoxygenase (5-LO). Here, we present a novel putative protein isoform of human 5-LO that lacks exon 4, termed 5-LOΔ4, identified in cells of lymphoid origin, namely the Burkitt lymphoma cell lines Raji and BL41 as well as primary B and T cells. Deletion of exon 4 does not shift the reading frame and therefore the mRNA is not subjected to non-mediated mRNA decay (NMD). By eliminating exon 4, the amino acids Trp144 until Ala184 are omitted in the corresponding protein. Transfection of HEK293T cells with a 5-LOΔ4 expression plasmid led to expression of the corresponding protein which suggests that the 5-LOΔ4 isoform is a stable protein in eukaryotic cells. We were also able to obtain soluble protein after expression in E. coli and purification. The isoform itself lacks canonical enzymatic activity as it misses the non-heme iron but it still retains ATP-binding affinity. Differential scanning fluorimetric analysis shows two transitions, corresponding to the two domains of 5-LO. Whilst the catalytic domain of 5-LO WT is destabilized by calcium, addition of calcium has no influence on the catalytic domain of 5-LOΔ4. Furthermore, we investigated the influence of 5-LOΔ4 on the activity of 5-LO WT and proved that it stimulates 5-LO product formation at low protein concentrations. Therefore regulation of 5-LO by its isoform 5-LOΔ4 might represent a novel mechanism of controlling the biosynthesis of lipid mediators.
Assuntos
Araquidonato 5-Lipoxigenase/metabolismo , Araquidonato 5-Lipoxigenase/química , Araquidonato 5-Lipoxigenase/isolamento & purificação , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Estabilidade Enzimática , Escherichia coli/metabolismo , Células HEK293 , Humanos , Ferro/metabolismo , Isoenzimas/química , Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , Modelos Moleculares , Proteínas Recombinantes/metabolismo , TemperaturaRESUMO
Human 5-lipoxygenase (5-LO) can form dimers as shown here via native gel electrophoresis, gel filtration chromatography and LILBID (laser induced liquid bead ion desorption) mass spectrometry. After glutathionylation of 5-LO by diamide/glutathione treatment, dimeric 5-LO was no longer detectable and 5-LO almost exclusively exists in the monomeric form which showed full catalytic activity. Incubation of 5-LO with diamide alone led to a disulfide-bridged dimer and to oligomer formation which displays a strongly reduced catalytic activity. The bioinformatic analysis of the 5-LO surface for putative protein-protein interaction domains and molecular modeling of the dimer interface suggests a head to tail orientation of the dimer which also explains the localization of previously reported ATP binding sites. This interface domain was confirmed by the observation that 5-LO dimer formation and inhibition of activity by diamide was largely prevented when four cysteines (C159S, C300S, C416S, C418S) in this domain were mutated to serines.
Assuntos
Araquidonato 5-Lipoxigenase/química , Multimerização Proteica , Araquidonato 5-Lipoxigenase/isolamento & purificação , Araquidonato 5-Lipoxigenase/metabolismo , Diamida/química , Eletroforese Capilar , Glutationa/química , Humanos , Espectrometria de Massas , Modelos Moleculares , Estrutura Quaternária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Terminalia chebula has an esteemed origin in Indian mythology; its fruits are used to treat many diseases such as digestive, diabetes, colic pain, chronic cough, sore throat, asthma, etc. AIM OF THE STUDY: The water or ethanolic extracts of the fruits were reported to have anti-oxidant, anti-inflammatory, anti-cancer and radio-protector properties. The present study is to isolate and identify the compounds that inhibit COX and 5-LOX, the key enzymes involved in inflammation and carcinogenesis. MATERIALS AND METHODS: The ethanolic extract of the fruits was fractionated by RP-HPLC and fractions were tested for enzyme inhibition activity against COX and 5-LOX. One of the fractionated compounds showed potent dual inhibition against COX and 5-LOX. It was identified as chebulagic acid by LC-MS, NMR and IR analyses. The chebulagic acid was also tested for anti-proliferative activity. RESULTS: Chebulagic acid showed potent COX-LOX dual inhibition activity with IC(50) values of 15+/-0.288, 0.92+/-0.011 and 2.1+/-0.057 microM for COX-1, COX-2 and 5-LOX respectively. It also showed anti-proliferative activity against HCT-15, COLO-205, MDA-MB-231, DU-145 and K562 cell lines. Further mechanistic studies on COLO-205 cells revealed induction of apoptosis by chebulagic acid. CONCLUSIONS: Chebulagic acid, a COX-2 and 5-LOX dual inhibitor isolated from the fruits of Terminalia chebula, induces apoptosis in COLO-205 cells.
Assuntos
Apoptose/efeitos dos fármacos , Benzopiranos/farmacologia , Inibidores de Ciclo-Oxigenase/farmacologia , Glucosídeos/farmacologia , Inibidores de Lipoxigenase/farmacologia , Terminalia/química , Antioxidantes/farmacologia , Araquidonato 5-Lipoxigenase/isolamento & purificação , Compostos de Bifenilo/química , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Inibidores de Ciclo-Oxigenase 2/farmacologia , Fragmentação do DNA/efeitos dos fármacos , Citometria de Fluxo , Sequestradores de Radicais Livres/química , Radicais Livres/química , Frutas/química , Humanos , Indicadores e Reagentes , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Picratos/química , Espectrofotometria InfravermelhoRESUMO
The role of allosteric effector--sodium dodecyl sulfate (SDS) in the lipoxygenase catalysis in micelle system has been studied. The effect of the stable hydrophobic bis-nitroxides, blocking the free radical transformation, on the oxidation of linoleic acid or linoleic alcohol by 5-lipoxygenase from potato tuber has been investigated. The inhibiting effect of nitroxide compounds on oxidation of linoleic acid or linoleic alcohol by 5-lipoxygenase depends on SDS concentration. The inhibition percentage is determined by the substrate nature and presence of allosteric effector. The presence of SDS did not lead to an appreciable change in the pKa values of ionogenic enzyme groups. The effect of SDS and micellar system on thermodynamic parameters for thermoinactivation of 5-lipoxygenase was studied. It was found that thermoinactivation rate constants and activation energy of enzyme thermoinactivation were increased in the presence of SDS. It is suggested that interaction of 5-lipoxygenase and allosteric effector--SDS intensifies the dissociation of radical intermediates from the active site of the enzyme. These findings are of physiological significance in the light of the lipoxygenase involvement in the membrane lipid peroxidation.
Assuntos
Araquidonato 5-Lipoxigenase/química , Dodecilsulfato de Sódio/química , Regulação Alostérica , Araquidonato 5-Lipoxigenase/isolamento & purificação , Peroxidação de Lipídeos , Lipídeos de Membrana/química , Solanum tuberosum/enzimologia , Especificidade por SubstratoRESUMO
Influence of anionogenic phospholipid of phosphatidic acid (PA) on oxidation of linoleic acid by 5-lipoxygenase (5-LO) from Solanum tuberosum was studied. The influence of PA was studied in micellar system which consisted of mixed micelles of linolenic acid (LK), Lubrol PX and different quantity of enzyme effector PA. The reaction was initiated by addition of 5-LO. It was established that 5-LO had two pHopt. in the presence of 50 microM phosphatidic acid: pH 5.0 and 6.9. In concentration of 50 microM PA was able to activate 5-LO 15 times at pH 5.0. The reaction maximum velocity (Vmax) coincided with Vmax of lipoxygenase reaction without the effector at pH 6.9 under such conditions. It was found that 30-50 microM phospholipid in the reaction mixture decreased the concentration of half saturation by the substrate by 43-67%. The enzyme demonstrated positive cooperation in respect of the substrate, the reaction is described by the Hill equation. Hill coefficient value (h) of the substrate was 3.34 +/- 0.22 (pH 6.9) and 5.61 +/- 0.88 (pH 5.0), that is with the change of pH to acidic region the number of substrate molecules increased and they could interact with the enzyme molecule. In case of substrate insufficiency the enzyme demonstrated positive cooperation of PA, it added from 4 to 3 effectors' molecules at pH 5.0, that is the phospholipid acted as the allosteric regulator of 5-LO. A comparative analysis of the influence of 4-hydroxy-TEMPO displayed, that the level of nonenzymatic processes in the case of physiological pH values was lower by 15-50% in the presence of PA in the range of 30-80 microM than without the effector.
Assuntos
Araquidonato 5-Lipoxigenase/química , Ácido Linoleico/química , Ácidos Fosfatídicos/química , Solanum tuberosum/enzimologia , Araquidonato 5-Lipoxigenase/isolamento & purificação , Catálise , Concentração de Íons de Hidrogênio , Cinética , Oxirredução , Espectrofotometria , Especificidade por SubstratoRESUMO
5-Lipoxygenase (5-LO), the key enzyme in leukotriene biosynthesis, is built of a catalytic C-terminal domain and a regulatory N-terminal C2-like domain. The C2-like domain is the target of many regulatory factors or proteins including Ca(2+), phospholipids, glycerides, coactosin-like protein and presumably other components that modulate the catalytic activity of 5-LO by acting at this domain, but the detailed underlying molecular mechanisms of these interactions are still unclear. In order to obtain the 5-LO C2-like domain as purified protein in good yields for further mechanistic studies and structure elucidation, a novel expression and purification approach has been applied. A plasmid was constructed expressing a fusion protein of maltose-binding protein (MBP) and the regulatory C2-like domain of 5-LO (AS 1-128), separated by a tobacco etch virus (TEV) protease-cleavage site. The fusion protein MBP-5LO1-128 could be essentially expressed as a soluble protein in Escherichia coli and was efficiently purified by amylose affinity chromatography. By means of this procedure, approximately 80mg purified fusion protein out of 1L E. coli culture were obtained. Digestion with TEV protease yielded the C2-like domain that was further purified using hydrophobic interaction chromatography. Alternatively, the uncleaved fusion protein MBP-5LO1-128 may be suitable to immobilize the C2-like domain on an amylose resin for co-factor interaction studies. Together, we present a convenient expression and purification strategy of the 5-LO C2-like domain that opens many possibilities for structural determination and mechanistic studies, aiming to reveal the precise role and function of this regulatory domain.
Assuntos
Araquidonato 5-Lipoxigenase/biossíntese , Estrutura Terciária de Proteína , Araquidonato 5-Lipoxigenase/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Endopeptidases/metabolismo , Humanos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/isolamento & purificaçãoRESUMO
Leukotrienes are important mediators in a number of inflammatory diseases and therefore are a target of several therapeutic approaches. The first committed step in the synthesis of leukotrienes is the conversion of arachidonic acid to leukotriene A(4) (LTA(4)) in two successive reactions catalyzed by 5-lipoxygenase (5-LOX). Assays to measure 5-LOX activity typically have been low throughput and time consuming. In this article, we describe a fluorescence assay that is amenable to high-throughput screening in a 384-well microplate format. The fluorescent signal is measured during oxidation of 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) by human 5-LOX. The assay has been found to reliably identify small molecule inhibitors of human 5-LOX. The IC(50) values of several 5-LOX inhibitors in this new assay are comparable to those determined in a standard spectrophotometric assay that measures the formation of the 5(S)-hydroperoxyeicosatetraenoic acid (5-HpETE) product. In addition, we demonstrate the use of the assay in a high-throughput screen of the Pfizer compound collection to identify inhibitors of 5-LOX.
Assuntos
Araquidonato 5-Lipoxigenase/isolamento & purificação , Inibidores de Lipoxigenase/análise , Inibidores de Lipoxigenase/química , Espectrofotometria Ultravioleta/métodos , Compostos Cromogênicos/química , Clonagem Molecular/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Fluoresceínas/química , Fluorescência , Corantes Fluorescentes/análise , Corantes Fluorescentes/química , Humanos , Indicadores e Reagentes , Concentração Inibidora 50 , Leucotrieno A4/química , Leucotrienos/química , Sensibilidade e Especificidade , Especificidade por SubstratoRESUMO
5-Lipoxygenase (5-LO) is the key enzyme in the biosynthesis of leukotrienes (LTs), biological mediators of host defense reactions and of inflammatory diseases. While the role of membrane binding in the regulation of 5-LO activity is well established, the effects of lipids on cellular activity when added to the medium has not been characterized. Here, we show such a novel function of the most abundant sulfated sterol in human blood, cholesterol sulfate (CS), to suppress LT production in human polymorphonuclear leukocytes (PMNL) and Mono Mac6 cells. We synthesized another anionic lipid, cholesterol phosphate, which demonstrated a similar capacity in suppression of LT synthesis in PMNL. Cholesteryl acetate was without effect. Cholesterol increased the effect of CS on 5-LO product synthesis. CS and cholesterol also inhibited arachidonic acid (AA) release from PMNL. Addition of exogenous AA increased the threshold concentration of CS required to inhibit LT synthesis. The effect of cholesterol and its anionic derivatives can arise from remodeling of the cell membrane, which interferes with 5-LO activation. The fact that cellular LT production is regulated by sulfated cholesterol highlights a possible regulatory role of sulfotransferases/sulfatases in 5-LO product synthesis.
Assuntos
Araquidonato 5-Lipoxigenase/metabolismo , Colesterol/análogos & derivados , Colesterol/farmacologia , Inibidores de Lipoxigenase , Neutrófilos/metabolismo , Ânions/farmacologia , Araquidonato 5-Lipoxigenase/isolamento & purificação , Ácido Araquidônico/antagonistas & inibidores , Calcimicina/farmacologia , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Clorpromazina/farmacologia , Ésteres do Colesterol/antagonistas & inibidores , Ésteres do Colesterol/farmacologia , Citocalasina D/farmacologia , Ativação Enzimática/efeitos dos fármacos , Humanos , Leucotrienos/biossíntese , Monócitos/efeitos dos fármacos , Monócitos/enzimologia , Monócitos/metabolismo , Neutrófilos/efeitos dos fármacos , Neutrófilos/enzimologiaRESUMO
The inhibiting effects of 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) and its 4-substituted derivatives in reactions of linoleyl acid or linoleyl alcohol oxidation catalyzed by potato tuber 5-lipoxygenase were investigated. Inhibiting properties of stable nitroxyl radicals in presence of lubrol and SDS were reduced at the transition from TEMPO to 4-hydroxy-TEMPO or 4-amino-TEMPO and increased at use of adamantane-1-carboxylic or 3-methyladamantane-1-carboxylic acid 1-oxyl-2,2,6,6-tetramethylpiperidine-4-yl esters. Enzyme activity at saturating concentrations of inhibitor was not suppressed completely, and decreased up to the certain level determined by the substrate nature. The dependence of partial inhibition efficiency on rotational correlation time of stable nitroxides in model micellar systems were analysed. It was supposed that 5-lipoxygenase inhibition includes the interaction of hydrophobic nitroxide with radical intermediate formed in enzymatic process.
Assuntos
Araquidonato 5-Lipoxigenase/química , Óxidos N-Cíclicos/química , Álcoois Graxos/química , Ácido Linoleico/química , Óxidos de Nitrogênio/química , Araquidonato 5-Lipoxigenase/isolamento & purificação , Catálise , Cinética , Estrutura Molecular , Oxirredução , Solanum tuberosum/químicaRESUMO
Mammalian 5-lipoxygenase (5-LO) catalyzes the conversion of arachidonic acid (AA) to leukotrienes, potent inflammatory mediators. 5-LO is activated by a Ca(2+)-mediated translocation to membranes, and demonstrates the characteristic features of interfacially activated enzymes, yet the mechanism of membrane binding of 5-LO is not well understood. In an attempt to understand the mechanism of lipid-mediated activation of 5-LO, we have studied the effects of a large set of lipids on human recombinant 5-LO activity, as well as mutual structural effects of 5-LO and membranes. In the presence of 0.35 mM phosphatidylcholine (PC) and 0.2 mM Ca(2+), there was substrate inhibition at >100 microM AA. Data analysis at low AA concentrations yielded the following: K(m) approximately 103 microM and k(cat) approximately 56 s(-1). 5-LO activity was supported by PC more than by any other lipid tested except for a cationic lipid, which was more stimulatory than PC. Binding of 5-LO to zwitterionic and acidic membranes was relatively weak; the extent of binding increased 4-8 times in the presence of Ca(2+), whereas binding to cationic membranes was stronger and essentially Ca(2+)-independent. Polarized attenuated total reflection infrared experiments implied that 5-LO binds to membranes at a defined orientation with the symmetry axis of the putative N-terminal beta-barrel tilted approximately 45 degrees from the membrane normal. Furthermore, membrane binding of 5-LO resulted in dehydration of the membrane surface and was paralleled with stabilization of the structures of both 5-LO and the membrane. Our results provide insight into the understanding of the effects of membrane surface properties on 5-LO-membrane interactions and the interfacial activation of 5-LO.
Assuntos
Araquidonato 5-Lipoxigenase/metabolismo , Lipídeos de Membrana/química , Araquidonato 5-Lipoxigenase/genética , Araquidonato 5-Lipoxigenase/isolamento & purificação , Cálcio/química , Cátions Bivalentes/química , Colesterol/química , Diglicerídeos/química , Ativação Enzimática/genética , Glicerídeos/química , Humanos , Membranas Intracelulares/química , Membranas Intracelulares/metabolismo , Cinética , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Lipídeos de Membrana/metabolismo , Modelos Químicos , Fosfatidilcolinas/química , Compostos de Amônio Quaternário/química , Espectroscopia de Infravermelho com Transformada de Fourier , Esfingolipídeos/química , Eletricidade Estática , Propriedades de SuperfícieRESUMO
Lipoxygenases are a group of non-heme iron dioxygenases which catalyze the formation of lipid hydroperoxides from unsaturated fatty acids. 5-Lipoxygenase (5LO) is of particular interest for formation of leukotrienes and lipoxins, implicated in inflammatory processes. In this study, electron paramagnetic resonance (EPR) spectroscopy was used to investigate the active site iron of purified recombinant human 5-lipoxygenase (5LO), and to explore the action of selenide on 5LO. After oxidation by lipid hydroperoxides, 5LO exhibited axial EPR spectra typified by a signal at g = 6.2. However, removal of the lipid hydroperoxides, their metabolites, and the solvent ethanol from the samples resulted in a shift to more rhombic EPR spectra (g = 5.17 and g = 9.0). Thus, many features of 5LO and soybean lipoxygenase-1 EPR spectra were similar, indicating similar flexible iron ligand arrangements in these lipoxygenases. Selenide (1.5 microM) showed a strong inhibitory effect on the enzyme activity of 5LO. In EPR, selenide abolished the signal at g = 6.2, typical for enzymatically active 5LO. Lipid hydroperoxide added to selenide-treated 5LO could not reinstate the signal at g = 6.2, indicating an irreversible change of the coordination of the active site iron.
Assuntos
Araquidonato 5-Lipoxigenase/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Inibidores de Lipoxigenase , Selênio/química , Araquidonato 5-Lipoxigenase/química , Araquidonato 5-Lipoxigenase/isolamento & purificação , Sítios de Ligação , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Ativação Enzimática , Etanol/química , Compostos Férricos/química , Compostos Férricos/metabolismo , Humanos , Ferro/química , Ferro/metabolismo , Peróxidos Lipídicos/química , Oxirredução , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
The positional specificity of arachidonic acid oxygenation is currently the decisive parameter for classification of lipoxygenases. Although the mechanistic basis of lipoxygenase specificity is not completely understood, sequence determinants for the positional specificity have been identified for various isoenzymes. In this study we altered the positional specificity of the human 5-lipoxygenase by multiple site-directed mutagenesis and assayed the leukotriene A(4) synthase activity of the mutant enzyme species with (5S,6E,8Z,11Z,14Z)-5-hydroperoxy-6,8,11,14-eicos atetraenoic acid (5S-HpETE) as substrate. The wild-type 5-lipoxygenase converts 5S-HpETE almost exclusively to leukotriene A(4) as indicated by the dominant formation of leukotriene A(4) hydrolysis products. Since leukotriene synthesis involves a hydrogen abstraction from C(10), it was anticipated that the 15-lipoxygenating quadruple mutant F359W + A424I + N425M + A603I might not exhibit a major leukotriene A(4) synthase activity. Surprisingly, we found that this quadruple mutant exhibited a similar leukotriene synthase activity as the wild-type enzyme in addition to its double oxygenation activity. The leukotriene synthase activity of the 8-lipoxygenating double mutant F359W + A424I was almost twice as high, and similar amounts of leukotriene A(4) hydrolysis products and double oxygenation derivatives were detected with this enzyme species. These data indicate that site-directed mutagenesis of the human 5-lipoxygenase that leads to alterations in the positional specificity favoring arachidonic acid 15-lipoxygenation does not suppress the leukotriene synthase activity of the enzyme. The residual 8-lipoxygease activity of the mutant enzyme and its augmented rate of 5-HpETE conversion may be discussed as major reasons for this unexpected result.
Assuntos
Araquidonato 5-Lipoxigenase/genética , Araquidonato 5-Lipoxigenase/metabolismo , Leucotrieno A4/metabolismo , Animais , Araquidonato 12-Lipoxigenase/metabolismo , Araquidonato 15-Lipoxigenase/metabolismo , Araquidonato 5-Lipoxigenase/isolamento & purificação , Catálise , Cromatografia Líquida de Alta Pressão , Ativação Enzimática/genética , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Leucotrienos/metabolismo , Mutagênese Sítio-Dirigida , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por Substrato/genéticaRESUMO
5-Lipoxygenase (5LO) catalyses the first two steps in the biosynthesis of leukotrienes, which are inflammatory mediators derived from arachidonic acid. 5LO activity is stimulated by ATP; however, a consensus ATP-binding site or nucleotide-binding site has not been found in its protein sequence. In the present study, affinity and photoaffinity labelling of 5LO with 5'-p-fluorosulphonylbenzoyladenosine (FSBA) and 2-azido-ATP showed that 5LO bound to the ATP analogues quantitatively and specifically and that the incorporation of either analogue inhibited ATP stimulation of 5LO activity. The stoichiometry of the labelling was 1.4 mol of FSBA/mol of 5LO (of which ATP competed with 1 mol/mol) or 0.94 mol of 2-azido-ATP/mol of 5LO (of which ATP competed with 0.77 mol/mol). Labelling with FSBA prevented further labelling with 2-azido-ATP, indicating that the same binding site was occupied by both analogues. Other nucleotides (ADP, AMP, GTP, CTP and UTP) also competed with 2-azido-ATP labelling, suggesting that the site was a general nucleotide-binding site rather than a strict ATP-binding site. Ca(2+), which also stimulates 5LO activity, had no effect on the labelling of the nucleotide-binding site. Digestion with trypsin and peptide sequencing showed that two fragments of 5LO were labelled by 2-azido-ATP. These fragments correspond to residues 73-83 (KYWLNDDWYLK, in single-letter amino acid code) and 193-209 (FMHMFQSSWNDFADFEK) in the 5LO sequence. Trp-75 and Trp-201 in these peptides were modified by the labelling, suggesting that they were immediately adjacent to the C-2 position of the adenine ring of ATP. Given the stoichiometry of the labelling, the two peptide sequences of 5LO were probably near each other in the enzyme's tertiary structure, composing or surrounding the ATP-binding site of 5LO.
Assuntos
Trifosfato de Adenosina/metabolismo , Araquidonato 5-Lipoxigenase/metabolismo , Marcadores de Afinidade , Sequência de Aminoácidos , Araquidonato 5-Lipoxigenase/química , Araquidonato 5-Lipoxigenase/isolamento & purificação , Sítios de Ligação , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Humanos , Cinética , Dados de Sequência Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Mg2+ gave dose-dependent activation of 5-lipoxygenase (5LO) in vitro. As for Ca2+, the activation depended on the presence of phosphatidylcholine (PC) vesicles, and the activation response was different at various combinations of arachidonate and PC. Stimulation of 5LO activity was observed with Mg2+ concentrations of 0.1-1 mM, similar to the concentration range of free Mg2+ in mammalian cells. However, to observe a clear increase in 5LO hydrophobicity, a higher concentration of Mg2+ (4 mM) was required, and at this concentration also 5LO activation was optimal. Combinations of Mg2+ with ATP (containing free Mg2+ and MgATP2- complex) gave better activation of 5LO than either agent alone. This effect of Mg2+ (and ATP) could be of interest in relation to basal 5LO activity in cells not subjected to a particular stimulus.
Assuntos
Araquidonato 5-Lipoxigenase/química , Ácidos Araquidônicos/química , Ativadores de Enzimas/química , Magnésio/química , Fosfatidilcolinas/química , Trifosfato de Adenosina/química , Trifosfato de Adenosina/farmacologia , Araquidonato 5-Lipoxigenase/genética , Araquidonato 5-Lipoxigenase/isolamento & purificação , Ácido Araquidônico/química , Cálcio/química , Cromatografia Líquida de Alta Pressão , Relação Dose-Resposta a Droga , Ativação Enzimática , Vetores Genéticos/metabolismo , Humanos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Água/químicaAssuntos
Araquidonato 5-Lipoxigenase/metabolismo , Proteínas Ativadoras de 5-Lipoxigenase , Animais , Araquidonato 5-Lipoxigenase/efeitos dos fármacos , Araquidonato 5-Lipoxigenase/isolamento & purificação , Proteínas de Transporte/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Citocinas/farmacologia , Indução Enzimática/efeitos dos fármacos , Humanos , Leucócitos/efeitos dos fármacos , Leucócitos/enzimologia , Proteínas de Membrana/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Microssomos/efeitos dos fármacos , Microssomos/enzimologia , Regulação para Cima/efeitos dos fármacosAssuntos
Araquidonato 5-Lipoxigenase/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Ativadoras de 5-Lipoxigenase , Animais , Araquidonato 5-Lipoxigenase/biossíntese , Araquidonato 5-Lipoxigenase/isolamento & purificação , Proteínas de Transporte/biossíntese , Proteínas de Transporte/isolamento & purificação , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Humanos , Cinética , Leucotrieno A4/metabolismo , Leucotrienos/metabolismo , Ácidos Linoleicos/metabolismo , Peróxidos Lipídicos/metabolismo , Proteínas de Membrana/biossíntese , Proteínas de Membrana/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Spodoptera , Especificidade por Substrato , TransfecçãoRESUMO
Human 5-lipoxygenase requires ATP as a stimulatory factor. At the two preferred concentrations of the free Ca2+, 0.02 microM with a resting cell and 20 microM with a stimulated cell, Scatchard analysis revealed that 5-lipoxygenase has one affinity ATP binding site with a Kd of 4.6 microM at the low Ca2+ concentration but has two affinity ATP binding sites with a higher Kd of 4.4 microM and a lower Kd of 14.5 microM at the high Ca2+ concentration. In contrast, in a Tween 20 reaction system, 5-lipoxygenase had similar activation coefficients for ATP at both Ca2+ concentrations; these were 12.7 microM at the low Ca2+ concentration and 12.0 microM at the high Ca2+ concentration. These results showed that 5-lipoxygenase has an ATP binding site and suggest that self-association of 5-lipoxygenase in 20 microM Ca2+ may affect ATP binding affinity as measured by Scatchard analysis.
Assuntos
Trifosfato de Adenosina/metabolismo , Araquidonato 5-Lipoxigenase/química , Araquidonato 5-Lipoxigenase/metabolismo , Nucleotídeos de Adenina/farmacologia , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Araquidonato 5-Lipoxigenase/isolamento & purificação , Sítios de Ligação , Humanos , Cinética , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Ribonucleotídeos/farmacologia , EspectrofotometriaRESUMO
5-Lipoxygenase (5-LO) and 5-lipoxygenase-activating protein (FLAP) are two key proteins involved in the synthesis of leukotrienes (LT) from arachidonic acid. Although both alveolar macrophages (AM) and peripheral blood leukocytes (PBL) produce large amounts of LT after activation, 5-LO translocates from a soluble pool to a particulate fraction upon activation of PBL, but is contained in the particulate fraction in AM irrespective of activation. We have therefore examined the subcellular localization of 5-LO in autologous human AM and PBL collected from normal donors. While immunogold electron microscopy demonstrated little 5-LO in resting PBL, resting AM exhibited abundant 5-LO epitopes in the euchromatin region of the nucleus. The presence of substantial quantities of 5-LO in the nucleus of resting AM was verified by cell fractionation and immunoblot analysis and by indirect immunofluorescence microscopy. In both AM and PBL activated by A23187, all of the observable 5-LO immunogold labeling was found associated with the nuclear envelope. In resting cells of both types, FLAP was predominantly associated with the nuclear envelope, and its localization was not affected by activation with A23187. The effects of MK-886, which binds to FLAP, were examined in ionophore-stimulated AM and PBL. Although MK-886 inhibited LT synthesis in both cell types, it failed to prevent the translocation of 5-LO to the nuclear envelope. These results indicate that the nuclear envelope is the site at which 5-LO interacts with FLAP and arachidonic acid to catalyze LT synthesis in activated AM as well as PBL, and that in resting AM the euchromatin region of the nucleus is the predominant source of the translocated enzyme. In addition, LT synthesis is a two-step process consisting of FLAP-independent translocation of 5-LO to the nuclear envelope followed by the FLAP-dependent activation of the enzyme.