Rabbit Reticulocyte 15-lipoxygenase Research Articles

Computational analysis of R and S isoforms of 12-Lipoxygenases: Homology modeling and docking studies

The present study is aimed at predicting human 12R-LOX structure by constructing a homology model. Based upon Blast results, rabbit reticulocyte 15-Lipoxygenase 1LOX (protein data bank) was considered as a template for homology modeling. The 3D model was generated with Modeler in InsightII and further refined using AMBER. Further to understand the relationship of protein structure with stereo specificity, a comparative analysis of 12R-LOX model was done with that of 12S-LOX homology model to identify differences in the binding site topology and interacting residues. The large insertion of 31-aa seen in 12R-LOX is located beyond the N-terminal barrel and is accommodated on the outside of the protein without disruption of the overall tertiary structure. The 31-aa region includes SH3 domain binding PXXP motif, seven prolines and five arginines. The docking of the substrate, arachidonic acid was also performed. Our results show that the Gly441 and substrate orientation within the active site play an important role in stereo specificity of 12R-LOX.

Inhibitory activity of salicylic acid on lipoxygenase-dependent lipid peroxidation

Background Since iron is essential for lipoxygenase activity and salicylic acid (SA) can interact with the metal, possible lipoxygenase inhibition by SA was investigated. Methods Kinetic spectrophotometric evaluation of enzymatic lipid peroxidation catalyzed by soybean lipoxygenase (SLO), rabbit reticulocyte 15-lipoxygenase (RR15-LOX), porcine leukocyte 12-lipoxygenase (PL12-LOX) and human recombinant 5-lipoxygenase (HR5-LOX) with and without SA. Results SA inhibited linoleic, arachidonic and docosahexaenoic acid or human lipoprotein peroxidation catalyzed by SLO with IC 50 of, respectively, 107, 153, 47 and 108 μM. Using the same substrates, SA inhibited RR15-LOX with IC 50 of, respectively, 49, 63, 27 and 51 μM. Further, arachidonic acid peroxidation catalyzed by PL12-LOX and HR5-LOX was inhibited by SA with IC 50 of 101 and 168 μM, respectively. Enzymatic inhibition was complete, reversible and non-competitive. Conceivably due to its lower hydrophobicity, aspirin was less effective, indicating acetylation-independent enzyme inhibition. SA and aspirin were ineffective peroxyl radical scavengers but readily reduced Fe 3+, i.e. FeCl 3, to Fe 2+, suggesting their capacity to reduce Fe 3+ at the enzyme active site. Indeed, similar to the catecholic redox inhibitor nordihydroguaiaretic acid, SA inhibited with the same efficiency both ferric and the native ferrous SLO form, indicating that these compounds reduce the active ferric enzyme leading to its inactivation. General significance SA can inhibit lipoxygenase-catalyzed lipid peroxidation at therapeutic concentrations, suggesting its possible inhibitory activity against enzymatic lipid peroxidation in the clinical setting.

Binding Investigation of Human 5-Lipoxygenase with Its Inhibitors by SPR Technology Correlating with Molecular Docking Simulation

The binding features of a series of 5-lipoxygenase (5-LOX) inhibitors (caffeic acid, NDGA, AA-861, CDC, esculetin, gossypol and phenidone) to human 5-LOX have been studied by using surface plasmon resonance biosensor (SPR) technology based Biacore 3000 and molecular docking simulation analyses. The SPR results showed that the equilibrium dissociation constant (KD) values evaluated by Biacore 3000 for the inhibitors showed a good correlation with its reported IC50, suggesting that SPR technology might be applicable as a direct assay method in screening new 5-LOX inhibitors at an early stage. In addition, the 3D structural model of 5-LOX was generated according to the crystal structure of rabbit reticulocyte 15-lipoxygenase, and the molecular docking simulation analyses revealed that the predicted binding free energies for the inhibitors correlated well with the KD values measured by SPR assay, which implies the correctness of the constructed 3D structural model of 5-LOX. This current work has potential for application in structure-based 5-LOX inhibitor discovery.

Essential structural profile of a dual functional inhibitor against cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX): Molecular docking and 3D-QSAR analyses on DHDMBF analogues

It is recently proposed that compounds with equal capabilities of inhibiting COX and 5-LOX, both are key enzymes involved in the arachidonic acid (AA) cascade, are expected to be safer non-steroidal anti-inflammatory drugs (NSAIDs). To dig out helpful information in designing dual functional inhibitors against the two enzymes, homology modeling, molecular dynamics (MD) simulations, automated docking, and 3D-QSAR analyses were performed in this study on 21 COX-2/5-LOX dual inhibitors, namely, 7- tert-butyl-2,3-dihydro-3,3-dimethylbenzofuran (DHDMBF) analogues. A 3D-model of 5-LOX was built based on the high-resolution X-ray structure of rabbit reticulocyte 15-lipoxygenase. Molecular docking was then applied to locate the binding orientations and conformations of DHDMBF analogues with COX-2 and 5-LOX, respectively, leading to highly predictive CoMFA models constructed on the basis of the binding conformations with q 2 values of 0.782 and 0.634 for COX-2 and 5-LOX, respectively. In addition, CoMFA field distributions were found in good agreement with the structural characteristics of the corresponding binding sites. Both the docking simulations and QSAR analyses suggest that new potent dual inhibitors should share a structural feature with a moderately bulky group at R 2 position and a rather negatively charged group around the position of the carbonyl group of DHDMBFs. Therefore, the final 3D-QSAR models and the information of the inhibitor–enzyme interaction should be useful in developing new NSAIDs as anti-inflammation drugs with favorable safety profile.

High-Level Expression of Rabbit 15-Lipoxygenase Induces Collapse of the Mitochondrial pH Gradient in Cell Culture

A critical step in the development of mammalian erythroblasts into mature red blood cells is the extrusion of the nucleus, followed by intracellular degradation of the remaining organelles. It has been hypothesized that the breakdown of cellular organelles in rabbit reticulocytes is initiated by 15-lipoxygenase. In vitro, the purified rabbit reticulocyte 15-lipoxygenase binds and permeabilizes organellar membranes, thereby releasing the lumenal contents of the organelle. Here, we demonstrate that ectopic expression of 15-lipoxygenase leads to the collapse of the mitochondrial pH gradient in nonerythroid cells, using a novel reporter of mitochondrial pH, mito-pHluorin. No change in mitochondrial pH was observed with a mutant of 15-lipoxygenase that lacks enzymatic activity. These data demonstrate that 15-lipoxygenase is capable of disrupting the pH gradient maintained by mitochondria in living cells without additional factors specific for red blood cell development.

Dihydrolipoic acid inhibits 15-lipoxygenase-dependent lipid peroxidation.

The potential antioxidant effects of the hydrophobic therapeutic agent lipoic acid (LA) and of its reduced form dihydrolipoic acid (DHLA) on the peroxidation of either linoleic acid or human non-HDL fraction catalyzed by soybean 15-lipoxygenase (SLO) and rabbit reticulocyte 15-lipoxygenase (RR15-LOX) were investigated. DHLA, but not LA, did inhibit SLO-dependent lipid peroxidation, showing an IC(50) of 15 microM with linoleic acid and 5 microM with the non-HDL fraction. In specific experiments performed with linoleic acid, inhibition of SLO activity by DHLA was irreversible and of a complete, noncompetitive type. In comparison with DHLA, the well-known lipoxygenase inhibitor nordihydroguaiaretic acid and the nonspecific iron reductant sodium dithionite inhibited SLO-dependent linoleic acid peroxidation with an IC(50) of 4 and 100 microM, respectively, while the hydrophilic thiol N-acetylcysteine, albeit possessing iron-reducing and radical-scavenging properties, was ineffective. Remarkably, DHLA, but not LA, was also able to inhibit the peroxidation of linoleic acid and of the non-HDL fraction catalyzed by RR15-LOX with an IC(50) of, respectively, 10 and 5 microM. Finally, DHLA, but once again not LA, could readily reduce simple ferric ions and scavenge efficiently the stable free radical 1,1-diphenyl-2-pycrylhydrazyl in ethanol; DHLA was considerably less effective against 2,2'-azobis(2-amidinopropane) dihydrochloride-mediated, peroxyl radical-induced non-HDL peroxidation, showing an IC(50) of 850 microM. Thus, DHLA, at therapeutically relevant concentrations, can counteract 15-lipoxygenase-dependent lipid peroxidation; this antioxidant effect may stem primarily from reduction of the active ferric 15-lipoxygenase form to the inactive ferrous state after DHLA-enzyme hydrophobic interaction and, possibly, from scavenging of fatty acid peroxyl radicals formed during lipoperoxidative processes. Inhibition of 15-lipoxygenase oxidative activity by DHLA could occur in the clinical setting, eventually resulting in specific antioxidant and antiatherogenic effects.

Effect of catechol derivatives on cell growth and lipoxygenase activity.

Derivatives of salicylic acid have been synthesized as potential lipoxygenase inhibitors. Agents containing a phenolic dihydroxy moiety showed potent (IC(50)10(-6)-10(-7) M) inhibition of the growth of murine colonic tumour cells in vitro, and were effective inhibitors of 5-, 12- and 15-lipoxygenase in intact cells. The catechols were also potent inhibitors of rabbit reticulocyte 15-lipoxygenase (IC(50) approximately 1 microM).

Specificity and inhibition by antioxidant of lipid peroxidation by lipoxygenase: effects of substrate, lipoxygenase and milieu

Although lipid peroxidation by lipoxygenases usually results in a site-, stereo- and enantio-specific mechanism, it was found that the oxidation of phospholipids incorporated into the liposomal membrane by soybean lipoxygenase, but not by rabbit reticulocyte 15-lipoxygenase, was proceeded entirely by a nonspecific mechanism. The radical-scavenging antioxidants such as α-tocopherol did not inhibit specific oxidation; however, they did suppress the nonspecific oxidation.

15-Lipoxygenase-1: A Prooxidant Enzyme

Human and rabbit reticulocyte 15-lipoxygenase (15-lipoxygenase-1) and the leukocyte-type 12-lipoxygenases (12/15-lipoxygenases) of pig, beef, mouse and rat constitute a particular subfamily of mammalian lipoxygenases (reticulocyte-type lipoxygenases) with unique properties and functions. They catalyze enzymatic lipid peroxidation in complex biological structures via direct dioxygenation of phospholipids and cholesterol esters of biomembranes and plasma lipoproteins. Moreover, they are a source of free radicals initiating non-enzymatic lipid peroxidation and other oxidative processes. Expression and activity of reticulocyte-type lipoxygenases are highly regulated. Moreover, the susceptibility of intracellular membranes toward these lipoxygenases is controlled and may be increased together with lipoxygenase activity under conditions of oxidative stress. Thus, oxidative stress may favor a concerted package of lipoxygenase-mediated enzymatic and non-enzymatic lipid peroxidation and co-oxidative processes. Reaction of reticulocyte-type lipoxygenases with low-density lipoprotein renders the latter atherogenic and appears to be involved in the formation of atherosclerotic lesions.

15-Lipoxygenation of leukotriene A 4: Studies of 12- and 15-lipoxygenase efficiency to catalyze lipoxin formation

The unstable epoxide leukotriene (LT) A 4 is a key intermediate in leukotriene biosynthesis, but may also be transformed to lipoxins via a second lipoxygenation at C-15. The capacity of various 12- and 15-lipoxygenases, including porcine leukocyte 12-lipoxygenase, a human recombinant platelet 12-lipoxygenase preparation, human platelet cytosolic fraction, rabbit reticulocyte 15-lipoxygenase, soybean 15-lipoxygenase and human eosinophil cytosolic fraction, to catalyze conversion of LTA 4 to lipoxins was investigated and standardized against the ability of the enzymes to transform arachidonic acid to 12- or 15-hydroxyeicosatetraenoic acids (HETE), respectively. The highest ratio between the capacity to produce lipoxins and HETE (LX/HETE ratio) was obtained for porcine leukocyte 12-lipoxygenase with an LX/HETE ratio of 0.3. In addition, the human platelet 100 000× g supernatant 12-lipoxygenase preparation and the human platelet recombinant 12-lipoxygenase and human eosinophil 100 000× g supernatant 15-lipoxygenase preparation possessed considerable capacity to produce lipoxins (ratio 0.07, 0.01 and 0.02 respectively). In contrast, lipoxin formation by the rabbit reticulocyte and soybean 15-lipoxygenases was much less pronounced (LX/HETE ratios <0.002). Kinetic studies of the human lipoxygenases revealed lower apparent K m for LTA 4 (9–27 μM), as compared to the other lipoxygenases tested (58–83 μM). The recombinant human 12-lipoxygenase demonstrated the lowest K m value for LTA 4 (9 μM) whereas the porcine leukocyte 12-lipoxygenase had the highest V max. The profile of products was identical, irrespective of the lipoxygenase used. Thus, LXA 4 and 6 S-LXA 4 together with the all- trans LXA 4 and LXB 4 isomers were isolated. Production of LXB 4 was not observed with any of the lipoxygenases. The lipoxygenase inhibitor cinnamyl-3,4-dihydroxy-α-cyanocinnamate was considerably more efficient to inhibit conversion of LTA 4 to lipoxins, as compared to the inhibitory effect on 12-HETE formation from arachidonic acid (IC 50 1 and 50 μM, respectively) in the human platelet cytosolic fraction.

15-Lipoxygenase Catalytically Consumes Nitric Oxide and Impairs Activation of Guanylate Cyclase

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.

Intra- and Extracellular Expression of Rabbit Reticulocyte 15-Lipoxygenase in the Baculovirus/Insect Cell System

Rabbit reticulocyte 15-lipoxygenase was expressed as intracellular enzyme and as export protein in High Five cells. While intracellular expression in the baculovirus/insect cell system was already reported for various mammalian lipoxygenases, we have developed a strategy for secretion of this cytosolic enzyme using the signal sequence of human interleukin-2. Expression levels of 10 mg/liter (intracellular strategy) and 18 mg/liter (extracellular strategy) were obtained. The recombinant enzyme expressed as intracellular protein was purified to apparent homogeneity by anion-exchange chromatography on a Mono-Q column with an overall recovery of 80% enzyme activity. For the final enzyme preparation, a specific linoleic acid oxygenase activity of 16.7 micromol 13-hydroperoxyoctadeca-9,11-dieic acid formation mg-1 min-1 was determined, which corresponds to a molecular turnover number of 21 s-1. Similar turnover rates have been reported for the native rabbit 15-lipoxygenase. Extracellularly expressed recombinant 15-lipoxygenase exhibited a heterogeneity in anion-exchange chromatography. Three major peaks of 15-lipoxygenase activity were eluted from a Mono-Q column and the relative amounts of these isoforms varied from batch to batch of enzyme expression. One of the major isoenzymes which cochromatographed with the native 15-lipoxygenase was purified to homogeneity from the cell-free culture supernatant and exhibited a specific activity of 5.1 micromol 13-hydroperoxyoctadeca-9,11-dienoic acid formation mg-1 min-1 (turnover rate of 6.1 s-1). The recombinant enzyme species were characterized with respect to their protein-chemical and enzymatic properties and no differences to the native rabbit 15-lipoxygenase were detected.

Inhibition of Mammalian 15-Lipoxygenase-Dependent Lipid Peroxidation in Low-Density Lipoprotein by Quercetin and Quercetin Monoglucosides

Lipoxygenase is suggested to be involved in the early event of atherosclerosis by inducing plasma low-density lipoprotein (LDL) oxidation in the subendothelial space of the arterial wall. Since flavonoids such as quercetin are recognized as lipoxygenase inhibitors and they occur mainly in the glycoside form, we assessed the effect of quercetin and its glycosides (quercetin 3-O-β-glucopyranoside, Q3G; quercetin 4′-O-β-glucopyranoside, Q4′G; quercetin 7-O-β-glucopyranoside, Q7G) on rabbit reticulocyte 15-lipoxygenase (15-Lox)-induced human LDL lipid peroxidation and compared it with the inhibition obtained by ascorbic acid and α-tocopherol, the main water-soluble and lipid-soluble antioxidants in blood plasma, respectively. Quercetin inhibited the formation of cholesteryl ester hydroperoxides (CE-OOH) and endogenous α-tocopherol consumption effectively throughout the incubation period of 6 h. Ascorbic acid exhibited an effective inhibition only in the initial stage and LDL preloaded with fivefold α-tocopherol did not affect the formation of CE-OOH compared with the native LDL. CE-OOH formation was inhibited by both quercetin and quercetin monoglucosides in a concentration-dependent manner. Quercetin, Q3G, and Q7G exhibited a higher inhibitory effect than Q4′G (IC50: 0.3–0.5 μM for quercetin, Q3G, and Q7G and 1.2 μM for Q4′G). While endogenous α-tocopherol was completely depleted after 2 h of LDL oxidation, quercetin, Q7G, and Q3G prevented the consumption of α-tocopherol. Quercetin and its monoglucosides were also exhausted during the LDL oxidation. These results indicate that quercetin glycosides as well as its aglycone are capable of inhibiting lipoxygenase-induced LDL oxidation more efficiently than ascorbic acid and α-tocopherol.

Membrane Translocation of 15-Lipoxygenase in Hematopoietic Cells Is Calcium-Dependent and Activates the Oxygenase Activity of the Enzyme

AbstractMammalian 15-lipoxygenases, which have been implicated in the differentiation of hematopoietic cells are commonly regarded as cytosolic enzymes. Studying the interaction of the purified rabbit reticulocyte 15-lipoxygenase with various types of biomembranes, we found that the enzyme binds to biomembranes when calcium is present in the incubation mixture. Under these conditions, an oxidation of the membrane lipids was observed. The membrane binding was reversible and led to an increase in the fatty acid oxygenase activity of the enzyme. To find out whether such a membrane binding also occurs in vivo, we investigated the intracellular localization of the enzyme in stimulated and resting hematopoietic cells by immunoelectron microscopy, cell fractionation studies and activity assays. In rabbit reticulocytes, the 15-lipoxygenase was localized in the cytosol, but also bound to intracellular membranes. This membrane binding was also reversible and the detection of specific lipoxygenase products in the membrane lipids indicated the in vivo activity of the enzyme on endogenous substrates. Immunoelectron microscopy showed that in interleukin-4 –treated monocytes, the 15-lipoxygenase was localized in the cytosol, but also at the inner side of the plasma membrane and at the cytosolic side of intracellular vesicles. Here again, cell fractionation studies confirmed the in vivo membrane binding of the enzyme. In human eosinophils, which constitutively express the 15-lipoxygenase, the membrane bound share of the enzyme was augmented when the cells were stimulated with calcium ionophore. Only under these conditions, specific lipoxygenase products were detected in the membrane lipids. These data suggest that in hematopoietic cells the cytosolic 15-lipoxygenase translocates reversibly to the cellular membranes. This translocation, which increases the fatty acid oxygenase activity of the enzyme, is calcium-dependent, but may not require a special docking protein.

Membrane Translocation of 15-Lipoxygenase in Hematopoietic Cells Is Calcium-Dependent and Activates the Oxygenase Activity of the Enzyme

Mammalian 15-lipoxygenases, which have been implicated in the differentiation of hematopoietic cells are commonly regarded as cytosolic enzymes. Studying the interaction of the purified rabbit reticulocyte 15-lipoxygenase with various types of biomembranes, we found that the enzyme binds to biomembranes when calcium is present in the incubation mixture. Under these conditions, an oxidation of the membrane lipids was observed. The membrane binding was reversible and led to an increase in the fatty acid oxygenase activity of the enzyme. To find out whether such a membrane binding also occurs in vivo, we investigated the intracellular localization of the enzyme in stimulated and resting hematopoietic cells by immunoelectron microscopy, cell fractionation studies and activity assays. In rabbit reticulocytes, the 15-lipoxygenase was localized in the cytosol, but also bound to intracellular membranes. This membrane binding was also reversible and the detection of specific lipoxygenase products in the membrane lipids indicated the in vivo activity of the enzyme on endogenous substrates. Immunoelectron microscopy showed that in interleukin-4 –treated monocytes, the 15-lipoxygenase was localized in the cytosol, but also at the inner side of the plasma membrane and at the cytosolic side of intracellular vesicles. Here again, cell fractionation studies confirmed the in vivo membrane binding of the enzyme. In human eosinophils, which constitutively express the 15-lipoxygenase, the membrane bound share of the enzyme was augmented when the cells were stimulated with calcium ionophore. Only under these conditions, specific lipoxygenase products were detected in the membrane lipids. These data suggest that in hematopoietic cells the cytosolic 15-lipoxygenase translocates reversibly to the cellular membranes. This translocation, which increases the fatty acid oxygenase activity of the enzyme, is calcium-dependent, but may not require a special docking protein.

Phenylalanine 353 is a Primary Determinant for the Positional Specificity of Mammalian 15-Lipoxygenases

Mammalian lipoxygenases are implicated in the biosynthesis of inflammatory mediators, in the pathogenesis of atherosclerosis and in the process of blood cell differentiation and maturation. With respect to their reaction specificity, three major types of mammalian lipoxygenases (15-lipoxygenases, 12-lipoxygenases and 5-lipoxygenases) may be classified. Although this nomenclature is commonly used, the mechanistic reasons for the positional specificity of lipoxygenases are not well understood. We investigated the structural reasons for lipoxygenase specificity by a combination of chimera formation and site-directed mutagenesis, and identified phenylalanine 353 as primary determinant for the positional specificity of rabbit reticulocyte 15-lipoxygenase. Modeling of the enzyme-substrate interaction suggested that the alignment of arachidonic acid at the active site appears to be influenced by this residue. According to the substrate orientation, the 15-lipoxygenase may be differentiated from two types of mammalian 12-lipoxygenases.

Reactivity of Mammalian 15-Lipoxygenase with Phospholipids in Large Unilamellar Liposomes

The reactivity of rabbit reticulocyte 15-lipoxygenase (15-LOX) with phosphatidylcholine (PC) possessing linoleic acid (LA-PC), arachidonic acid (AA-PC), or docosahexaenoic acid (DHA-PC) was investigated by measuring oxygen uptake in the suspension of large unilamellar liposomes (LUV). The 15-LOX reaction with PC LUV was found to follow Michaelis-Menten kinetics. The apparent values of the Michaelis constants (Km) of LUV of LA-PC, AA-PC, and DHA-PC were nearly the same (1.0-1.2 mM). However, the maximum velocity (Vmax) of DHA-PC was obviously two to three times lower than those of LA-PC and AA-PC. On the other hand, the oxidizability of PC perbis-allylic H of unsaturated fatty acids in the free radical chain reaction of LUV decreased with increasing degree of unsaturation, that is, LA-PC>AA-PC>DHA-PC. These results suggested that the oxidizability of the DHA moiety incorporated into biomembranes is not necessarily high in lipoxygenase reaction as in free radical chain reaction.

High rates of extracellular superoxide generation by cultured human fibroblasts: involvement of a lipid-metabolizing enzyme.

Expression of NADPH oxidase and low superoxide generation (approx. 0.06 nmol/min per 10(6) cells) by cytokine- or ionophore-stimulated human fibroblasts is known. However, we here show that these cells also contain an ectoplasmic enzyme, distinct from NADPH oxidase, which can generate superoxide (2.19 +/- 0.14 nmol/min per 10(6) cells) at levels similar to phorbol ester-stimulated monocytes on exogenous NADH addition. Superoxide generation was temperature-dependent, insensitive to chelation (desferal), and had a K(m) (app)(NADH) of 11.5 microM. Inhibitor studies showed that there was no involvement of NADPH oxidase (diphenylene iodonium, diphenyl iodonium), prostaglandin H synthase (indomethacin), xanthine oxidase (allopurinol), cytochrome P-450 (metyrapone) or mitochondrial respiration (rotenone, antimycin A). NAD+ was a competitive inhibitor, whereas NADPH supported 40% of the rate seen with NADH. No luminescence was observed after the addition of lactate, malate, pyruvate, GSH or L-cysteine. NADH-stimulated superoxide generation was enhanced by the addition of (3-30 microM) arachidonic acid, linoleic acid or (5S)-hydroxyeicosatetraenoic acid [(5S)-HETE] but not palmitic acid, (15S)-hydroperoxyeicosatetraenoic acid [(15S)-HPETE], (15S)-HETE or (12S)-HETE. Several features suggest involvement of an enzyme related to 15-lipoxygenase, and, in support of this, we show superoxide generation and NADH oxidation by recombinant rabbit reticulocyte 15-lipoxygenase. The large amounts of superoxide measured suggest that the fibroblast extracellular enzyme could be a major source of reactive oxygen species after tissue damage.

Bacterial expression, purification and partial characterization of recombinant rabbit reticulocyte 15-lipoxygenase

The recombinant rabbit reticulocyte 15-lipoxygenase has been expressed in E coli with a yield of about 50–70 βg pure lipoxygenase protein per l of liquid culture. The enzyme has been purified to apparent homogeneity from the bacteria lysis supernatant by ammonium sulfate precipitation, and two consecutive steps of anion exchange chromatography on a Mono Q column. As the native enzyme the recombinant lipoxygenase has a molecular mass of 75 kDa, an isoelectric point of 5.5 and oxygenates both linoleic acid (formation of 13 S-hydroperoxy-9 Z,13 E-octadecadienoic acid) and arachidonic acid. With the latter substrate it exhibits a dual positional specificity (formation of 15 S-hydroperoxy-5 Z,8 Z,11 Z,13 E-eicosatetraenoic acid and 12 S-hydroperoxy-5 Z,8 Z,10 E,14 Z-eicosatetraenoic acid in a ratio of 12:1). Furthermore, the enzyme is capable of oxygenating biomembranes, as indicated by HPLC analysis of esterified oxygenated polyenoic fatty acids.

Purification and crystallization of 15-lipoxygenase from rabbit reticulocytes

We report a new purification of rabbit reticulocyte 15-lipoxygenase that has resulted in the first crystallization of a mammalian lipoxygenase. The enzyme was purified to homogeneity (greater than 98% pure by SDS-PAGE) using high pressure liquid chromatography on hydrophobic-interaction, hydroxyapatite and cation-exchange columns. Crystals were grown by the vapor diffusion method from concentrated solutions of the protein in sodium phosphate buffer, pH 7.0. The hexagonal, rod-shaped crystals were on average 0.09 mm x 0.09 mm x 0.4 mm, with approximate unit cell dimensions of a = b = 260 A, c = 145 A. The crystals diffract to 5 A resolution.