Human Leukocyte 5-lipoxygenase Research Articles

5 S,15 S-Dihydroperoxyeicosatetraenoic Acid (5,15-diHpETE) as a Lipoxin Intermediate: Reactivity and Kinetics with Human Leukocyte 5-Lipoxygenase, Platelet 12-Lipoxygenase, and Reticulocyte 15-Lipoxygenase-1.

The reaction of 5 S,15 S-dihydroperoxyeicosatetraenoic acid (5,15-diHpETE) with human 5-lipoxygenase (LOX), human platelet 12-LOX, and human reticulocyte 15-LOX-1 was investigated to determine the reactivity and relative rates of producing lipoxins (LXs). 5-LOX does not react with 5,15-diHpETE, although it can produce LXA4 when 15-HpETE is the substrate. In contrast, both 12-LOX and 15-LOX-1 react with 5,15-diHpETE, forming specifically LXB4. For 12-LOX and 5,15-diHpETE, the kinetic parameters are kcat = 0.17 s-1 and kcat/ KM = 0.011 μM-1 s-1 [106- and 1600-fold lower than those for 12-LOX oxygenation of arachidonic acid (AA), respectively]. On the other hand, for 15-LOX-1 the equivalent parameters are kcat = 4.6 s-1 and kcat/ KM = 0.21 μM-1 s-1 (3-fold higher and similar to those for 12-HpETE formation by 15-LOX-1 from AA, respectively). This contrasts with the complete lack of reaction of 15-LOX-2 with 5,15-diHpETE [Green, A. R., et al. (2016) Biochemistry 55, 2832-2840]. Our data indicate that 12-LOX is markedly inferior to 15-LOX-1 in catalyzing the production of LXB4 from 5,15-diHpETE. Platelet aggregation was inhibited by the addition of 5,15-diHpETE, with an IC50 of 1.3 μM; however, LXB4 did not significantly inhibit collagen-mediated platelet activation up to 10 μM. In summary, LXB4 is the primary product of 12-LOX and 15-LOX-1 catalysis, if 5,15-diHpETE is the substrate, with 15-LOX-1 being 20-fold more efficient than 12-LOX. LXA4 is the primary product with 5-LOX but only if 15-HpETE is the substrate. Approximately equal proportions of LXA4 and LXB4 are produced by 12-LOX but only if LTA4 is the substrate, as described previously [Sheppard, K. A., et al. (1992) Biochim. Biophys. Acta 1133, 223-234].

Endogenous metabolites of vitamin E limit inflammation by targeting 5-lipoxygenase

Systemic vitamin E metabolites have been proposed as signaling molecules, but their physiological role is unknown. Here we show, by library screening of potential human vitamin E metabolites, that long-chain ω-carboxylates are potent allosteric inhibitors of 5-lipoxygenase, a key enzyme in the biosynthesis of chemoattractant and vasoactive leukotrienes. 13-((2R)-6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)-2,6,10-trimethyltridecanoic acid (α-T-13′-COOH) can be synthesized from α-tocopherol in a human liver-on-chip, and is detected in human and mouse plasma at concentrations (8–49 nM) that inhibit 5-lipoxygenase in human leukocytes. α-T-13′-COOH accumulates in immune cells and inflamed murine exudates, selectively inhibits the biosynthesis of 5-lipoxygenase-derived lipid mediators in vitro and in vivo, and efficiently suppresses inflammation and bronchial hyper-reactivity in mouse models of peritonitis and asthma. Together, our data suggest that the immune regulatory and anti-inflammatory functions of α-tocopherol depend on its endogenous metabolite α-T-13′-COOH, potentially through inhibiting 5-lipoxygenase in immune cells.

Novel series of benzoquinones with high potency against 5-lipoxygenase in human polymorphonuclear leukocytes

5-Lipoxygenase (5-LO) is a potential target for pharmacological intervention with various inflammatory and allergic diseases. Starting from the natural dual 5-LO/microsomal prostaglandin E2 synthase (mPGES)-1 inhibitor embelin (2,5-dihydroxy-3-undecyl-1,4-benzoquinone, 2) that suppresses 5-LO activity in human primary leukocytes with IC50 = 0.8–2 μM, we synthesized 48 systematically modified derivatives of 2. We modified the 1,4-quinone to 1,2-quinone, mono- or bimethylated the hydroxyl groups, and varied the C11-n-alkyl residue (C4- to C16-n-alkyl or prenyl) of 2. Biological evaluation yields potent analogues being superior over 2 and obvious structure-activity relationships (SAR) for inhibition of 5-LO. Interestingly, conversion to 1,2-benzoquinone and bimethylation of the hydroxyl moieties strongly improves 5-LO inhibition in polymorphonuclear leukocytes versus 2 up to 60-fold, exemplified by the C12-n-alkyl derivative 22c (4,5-dimethoxy-3-dodecyl-1,2-benzoquinone) with IC50 = 29 nM. Regarding inhibition of mPGES-1, none of the novel benzoquinones could outperform the parental compound 2 (IC50 = 0.21 μM), and only modest suppressive effects on 12- and 15-LOs were evident. Together, our detailed SAR study reveals 22c as highly potent 5-LO-selective lead compound in intact cells that warrants further preclinical evaluation as anti-inflammatory agent.

Identification and absolute configuration of dihydroxy-arachidonic acids formed by oxygenation of 5S-HETE by native and aspirin-acetylated COX-2

Biosynthesis of the prostaglandin endoperoxide by the cyclooxygenase (COX) enzymes is accompanied by formation of a small amount of 11R-hydroxyeicosatetraenoic acid (HETE), 15R-HETE, and 15S-HETE as by-products. Acetylation of COX-2 by aspirin abrogates prostaglandin synthesis and triggers formation of 15R-HETE as the sole product of oxygenation of arachidonic acid. Here, we investigated the formation of by-products of the transformation of 5S-HETE by native COX-2 and by aspirin-acetylated COX-2 using HPLC-ultraviolet, GC-MS, and LC-MS analysis. 5S,15S- dihydroxy (di)HETE, 5S,15R-diHETE, and 5S,11R-diHETE were identified as by-products of native COX-2, in addition to the previously described di-endoperoxide (5S,15S-dihydroxy-9S,11R,8S,12S-diperoxy-6E,13E-eicosadienoic acid) as the major oxygenation product. 5S,15R-diHETE was the only product formed by aspirin-acetylated COX-2. Both 5,15-diHETE and 5,11-diHETE were detected in CT26 mouse colon carcinoma cells as well as in lipopolysaccharide-activated RAW264.7 cells incubated with 5S-HETE, and their formation was attenuated in the presence of the COX-2 specific inhibitor, NS-398. Aspirin-treated CT26 cells gave 5,15-diHETE as the most prominent product formed from 5S-HETE. 5S,15S-diHETE has been described as a product of the cross-over of 5-lipoxygenase (5-LOX) and 15-LOX activities in elicited rat mononuclear cells and human leukocytes, and our studies implicate cross-over of the 5-LOX and COX-2 pathways as an additional biosynthetic route.

Resolvin E2: Identification and Anti-Inflammatory Actions: Pivotal Role of Human 5-Lipoxygenase in Resolvin E Series Biosynthesis

The family of resolvins consists of omega-3 fatty acid-derived mediators, including E series resolvins generated from eicosapentaenoic acid (EPA), and carry potent anti-inflammatory properties. Here, we report the isolation, identification, and bioactions of resolvin E2 (RvE2), which is 5S,18-dihydroxy-eicosapentaenoic acid. RvE2 stopped zymosan-induced polymorphonuclear (PMN) leukocyte infiltration and displayed potent anti-inflammatory properties in murine peritonitis. We also demonstrate that human recombinant 5-lipoxygenase generates RvE2 from a common precursor of E series resolvins, namely, 18-hydroxyeicosapentaenoate (18-HEPE). Furthermore, the initial 5-hydroperoxide intermediate was also converted to a 5(6)-epoxide intermediate in RvE1 formation. These results demonstrate that RvE2, together with RvE1, may contribute to the beneficial actions of omega-3 fatty acids in human diseases. Moreover, they indicate that the 5-lipoxygenase in human leukocytes is a pivotal enzyme that can produce both pro- and anti-inflammatory chemical mediators.

Inhibition of Human 5-Lipoxygenase and Anti-Neoplastic Effects by 2-Amino-1,4-Benzoquinones

We have recently presented the synthesis of 2-amino-1,4-benzoquinones by nuclear amination of p-hydroquinones with primary aromatic amines using fungal laccases as catalysts. In the present report, a series of selected 2-amino-1,4-benzoquinones was tested for biological activities, such as inhibition of human 5-lipoxygenase and anti-proliferative/anti-neoplastic effects. Compound 9 (2-[4'-(iso-propylphenyl)-amino]-5,6-dimethyl-1,4-benzoquinone) was identified as the most potent aminoquinone derivative, suppressing 5-lipoxygenase in intact human polymorphonuclear leukocytes as well as in crude enzyme preparations in the low micromolar range (IC50 = 6 microM). Structure-activity relationships are discussed. Of interest, the 5-lipoxygenase inhibitory properties of 2-amino-1,4-benzoquinones in intact cells correlated to the anti-neoplastic activities of the compounds in breast and urinary bladder cancer cell lines. Based on these features, bioactive 2-amino-1,4-benzoquinones may possess potential for the pharmacological treatment of diseases associated with elevated 5-lipoxygenase activity, in particular certain types of cancer.

Hypertonicity suppresses ionophore-induced product formation and translocation of 5-lipoxygenase in human leukocytes

5-Lipoxygenase (5-LO) initiates the biosynthesis of proinflammatory leukotrienes from arachidonic acid (AA). Here, we demonstrate that hypertonicity suppresses ionophore-induced 5-LO product formation reversibly in isolated human polymorphonuclear leukocytes (PMNL) and in Mono Mac 6 cells. Hypertonicity blocked the liberation of AA and abrogated translocation of 5-LO to the nuclear membrane. Accordingly, in the presence of exogenous AA, 5-LO product formation was less affected. The effects of hypertonicity were a result of cell shrinkage and not cytosolic hyperosmolarity. Hypertonicity did not inhibit the rapid increase in intracellular Ca(2)(+) induced by ionophores but prevented the ionophore-induced activation of p38 MAPK-regulated MAPKAP kinases, which can phosphorylate and activate 5-LO (and cPLA(2)). In summary, we show that hypertonicity blocks agonist-induced release of AA, 5-LO product formation, and translocation and in parallel, prevents activation of p38 MAPK and downstream 5-LO kinases in leukocytes.

Molecular cloning of a 12-lipoxygenase cDNA from rat brain.

A cDNA encoding an arachidonate 12-lipoxygenase from rat brain was obtained by polymerase chain reaction cloning. Primers specific for porcine leukocyte 12-lipoxygenase cDNA were used to isolate the initial polymerase-chain-reaction product (395 bp). The final sequence of the rat 12-lipoxygenase cDNA coding region (1989 bp) was verified by analysis of several separate polymerase-chain-reaction products. The open reading frame corresponded to a protein of 662 amino acid residues, with a calculated molecular mass of 75,305 Da. Also the rat 12-lipoxygenase contained the six conserved histidines, characteristic for all cloned lipoxygenases. It displayed the highest degree of identity to porcine leukocyte 12-lipoxygenase (71%) and to human 15-lipoxygenase (75%), with less resemblance to human platelet 12-lipoxygenase (59%) or rat leukocyte 5-lipoxygenase (41%). The recombinant enzyme was expressed in Escherichia coli and incubated with arachidonic acid. Primarily 12-lipoxygenase (but also some 15-lipoxygenase) enzyme activity was obtained. A part of the brain 12-lipoxygenase cDNA was used as probe in Northern blots. A 2.7-kb mRNA was more abundant in RNA from rat leukocytes, lung, and aorta, than in RNA from rat brain. Sequencing of parts of the corresponding cDNAs (from leukocytes and lung), and comparison to the brain 12-lipoxygenase sequence, indicated that these mRNAs from the different rat tissues were identical.

On the expression and regulation of 5-lipoxygenase in human lymphocytes.

The expression of arachidonate 5-lipoxygenase (arachidonate:oxygen 5-oxidoreductase, EC 1.13.11.34) and the 5-lipoxygenase-activating protein (FLAP) genes in human tonsillar B cells and lymphoblastoid B-cell lines was demonstrated at the transcriptional level by reverse transcription-PCR analysis. Also, five lymphoblastoid T-cell lines were investigated and found to express the FLAP gene but not the 5-lipoxygenase gene, suggesting that the transcriptional regulation of these two genes is different. Western blot analysis of the cytosolic proteins from a lymphoblastoid B-cell line with an antiserum raised against purified human leukocyte 5-lipoxygenase revealed an immunoreactive band that comigrated with recombinant human 5-lipoxygenase. Intact B cells produced very low amounts of leukotriene B4 and 5-hydroxyeicosatetraenoic acid upon stimulation with the calcium ionophore A23187 and arachidonic acid, in comparison to the amounts formed by sonicates of these cells. However, preincubation of intact lymphoblastoid B cells with the glutathione-depleting agents azodicarboxylic acid bis(dimethylamide) or 1-chloro-2,4-dinitrobenzene prior to the addition of the calcium ionophore A23187 and arachidonic acid led to similar amounts of leukotriene B4 as were formed by sonicated cells. In contrast, the glutathione synthesis inhibitor buthionine sulfoximine diminished the cellular level of glutathione by greater than 90% but did not influence the production of leukotriene B4 or 5-hydroxyeicosatetraenoic acid in intact cells. These results demonstrate that certain drugs affecting the redox status can stimulate the cryptic 5-lipoxygenase activity in intact lymphoblastoid B cells but that the mechanism of this activation is unclear and appears not to be directly related to intracellular glutathione levels.

Characterization of monoclonal antibodies against human leukocyte 5-lipoxygenase

Four mouse monoclonal IgG1 antibody-producing cell lines (5LO-1, 5LO-2, 5LO-3, 5LO-4), produced against highly purified human leukocyte 5-lipoxygenase have been characterized. The monoclonal antibodies produced by these cell lines exhibited differential reactivity against 5-lipoxygenase as determined by ELISA and immunoprecipitation analyses. Monoclonal antibodies 5LO-2 and 5LO-3 inhibited the activity of recombinant human leukocyte 5-lipoxygenase in a dose-dependent manner. This inhibition was selective for 5-lipoxygenase activity since these monoclonal antibodies did not inhibit human leukocyte 15-lipoxygenase or porcine leukocyte 12-lipoxygenase.

Inhibition of human leukocyte 5-lipoxygenase by a 4-hydroxybenzofuran, L-656,224: Evidence for enzyme reduction and inhibitor degradation

Detailed studies of the interaction of L-656,224 (2-[(4′-methoxyphenyl)methyl]-3-methyl-4-hydroxy-5-propyl-7-chlorobenzofuran) with 5-lipoxygenase were conducted using the enzymes from human and pig leukocytes. L-656,224 was a potent inhibitor of these 5-lipoxygenases although its efficiency varied with enzyme concentration. L-656,224 also stimulated the pseudoperoxidase activity of 5-lipoxygenase as measured by the consumption of 13-hydroperoxy-9,11-octadecadienoic acid (13-HPOD), indicating that this compound can reduce the enzyme. Furthermore the inhibitor was degraded rapidly by both cell-free leukocyte extracts and purified 5-lipoxygenase after incubation with 13-HPOD, ATP and calcium ions. The degradation of L-656,224 was also observed during inhibition of the lipoxygenase reaction and occurred mainly after the initial lag phase of the reaction when hydroperoxides begin to accumulate. A single major radioactive product was formed after incubation of [ 3H]L-656,224 with purified 5-lipoxygenase in the presence of 13-HPOD. This product was unstable and could not be isolated. During the course of the pseudoperoxidase reaction, [ 3H]L-656,224 covalently labelled the enzyme, suggesting that a chemically reactive species had been formed. These data are consistent with the hypothesis that L-656,224 reduces the oxidized form of the 5-lipoxygenase to an inactive form, with degradation of the inhibitor and regeneration of the active enzyme with hydroperoxides.

Cloning of the cDNA for human 12-lipoxygenase.

A full-length cDNA clone encoding 12-lipoxygenase (arachidonate:oxygen 12-oxidoreductase, EC 1.13.11.31) was isolated from a human platelet cDNA library by using a cDNA for human reticulocyte 15-lipoxygenase as probe for the initial screening. The cDNA had an open reading frame encoding 662 amino acid residues with a calculated molecular weight of 75,590. Three independent clones revealed minor heterogeneities in their DNA sequences. Thus, in three positions of the deduced amino acid sequence, there is a choice between two different amino acids. The deduced sequence from the clone plT3 showed 65% identity with human reticulocyte 15-lipoxygenase and 42% identity with human leukocyte 5-lipoxygenase. The 12-lipoxygenase cDNA recognized a 3.0-kilobase mRNA species in platelets and human erythroleukemia cells (HEL cells). Phorbol 12-tetradecanoyl 13-acetate induced megakaryocytic differentiation of HEL cells and 12-lipoxygenase activity and increased mRNA for 12-lipoxygenase. The identity of the cloned 12-lipoxygenase was assured by expression in a mammalian cell line (COS cells). Human platelet 12-lipoxygenase has been difficult to purify to homogeneity. The cloning of this cDNA will increase the possibilities to elucidate the structure and function of this enzyme.

Cloning and sequence analysis of the cDNA for arachidonate 12-lipoxygenase of porcine leukocytes.

The complete amino acid sequence of arachidonate 12-lipoxygenase (EC 1.13.11.31) of porcine leukocytes was deduced by cloning and sequence analysis of DNA complementary to its mRNA. The sequence was confirmed by automated Edman degradation of the N-terminal regions of the native enzyme and its proteolytic fragments. The cDNA had an open reading frame encoding 662 amino acid residues with a calculated molecular weight of 74,911. Amino acid residues 533-545, Cys-(Xaa)3-Cys-(Xaa)3-His-(Xaa)3-His, showed significant homology to the short cysteine- or histidine-containing sequences proposed as the metal-binding domains of transcription factors and various metal-containing proteins [Berg, J. M. (1986) Science 232, 485-487]. The amino acid sequence of 12-lipoxygenase exhibited 86% identity with human reticulocyte 15-lipoxygenase and showed 41% identity with human leukocyte 5-lipoxygenase. The 12-lipoxygenase cDNA recognized a 3.4-kilobase mRNA species in various porcine cell types, with the largest amount in leukocytes, followed by pituitary, lung, jejunum, and spleen.

Expression of cloned human reticulocyte 15-lipoxygenase and immunological evidence that 15-lipoxygenases of different cell types are related.

Cloned 15-lipoxygenase has been expressed for the first time in eukaryotic and prokaryotic cells. Transfection of osteosarcoma cells with a mammalian expression plasmid containing the cDNA for human reticulocyte 15-lipoxygenase resulted in cell lines that were capable of oxidizing body arachidonic acid and linoleic acid. The lipoxygenase metabolites were identified by reverse-phase and straight-phase high pressure liquid chromatography, ultraviolet spectroscopy, and direct mass spectrometry, verifying that the cDNA for 15-lipoxygenase encodes an enzyme with authentic 15-lipoxygenase activity. Incubation of the transformed cells with arachidonic acid generated 15-hydroxyeicosatetraenoic acid (HETE) and 12-HETE in a ratio of 8.6 to 1, demonstrating that 15-lipoxygenase can also perform 12-lipoxygenation. Lesser amounts of 15-keto-ETE, four isomers of 8,15-diHETE, and one isomer of 14,15-diHETE were observed. Incubation with linoleic acid generated predominantly 13-hydroxy linoleic acid. The reaction was inhibited by eicosatetraynoic acid but not by indomethacin. Antibodies to a peptide corresponding to a unique region of the predicted amino acid sequence were generated and shown to react with one major band of 70 kDa on immunoblots of human leukocyte 15-lipoxygenase. To obtain antibodies to the full length enzyme, the cDNA was subcloned into a bacterial expression vector and was expressed as a fusion with the CheY protein. The overexpressed protein was readily purified from bacteria and was shown to be immunoreactive to the peptide-derived antibody. Antibodies raised to this recombinant enzyme did not cross-react with human leukocyte 5-lipoxygenase but did identify 15-lipoxygenase in rabbit reticulocytes, human leukocytes, and tracheal epithelial cells, suggesting that the 15-lipoxygenases from these different cell types are structurally related.

Monohydroxylated fatty acid substrate specificity of human leukocyte 5-lipoxygenase and omega-hydroxylase.

Various monohydroxylated fatty acids were synthesized from eicosapolyenoic acids, namely arachidonic (20:4 omega-6), timnodonic (20:5 omega-3), dihomogammalinolenic (20:3 omega-6) and mead (20:3 omega-9) acids. 12-Hydroxy derivatives, as well as 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT), were produced with platelets as the enzyme source, and 15-hydroxy derivatives were produced by soya bean lipoxygenase treatment. Each monohydroxylated fatty acid was incubated with human leukocytes in the presence or absence of the calcium ionophore A23187, and dihydroxylated products were analysed by h.p.l.c. 12-Hydroxy derivatives of 20:4 omega-6, 20:5 omega-3 and 20:3 omega-9 were similarly oxygenated by both the 5-lipoxygenase and the omega-hydroxylase. As expected, the 12-hydroxy derivative of 20:3 omega-6 was not a substrate for 5-lipoxygenase, but surprisingly, omega-6 oxygenated products, like 15-OH-20:4 or HHT, were not converted by the enzyme, although being potential substrates because of the presence of two double bonds at C-5 and C-8. omega-6 oxygenated derivatives were also poorly converted by leukotriene B4 omega-hydroxylase, a cytochrome P-450-dependent enzyme. It is concluded that both leukocyte 5-lipoxygenase and omega-hydroxylase exhibit a substrate specificity towards monohydroxylated fatty acids with respect to their double bonds and/or the carbon position of the alcohol function.

Inhibition of human leukocyte 5-lipoxygenase by 15-HPETE and related eicosanoids

The inhibition of human leukocyte 5-lipoxygenase by 15-hydroperoxyeicosatetraenoic acid and its chemical or enzymatic rearrangement products was investigated. 15-Hydroperoxyeicosatetraenoic acid was the most potent inhibitor tested. The inhibition was found to be time dependent and is not due to chemical or enzymatic decomposition products nor metabolism of 15-hydroperoxyeicosatetraenoic acid to 5,15-dihydroperoxyeicosatetraenoic acid.

Characterization of cloned human leukocyte 5-lipoxygenase expressed in mammalian cells.

5-Lipoxygenase has been expressed in a mammalian osteosarcoma cell line transfected with the cloned cDNA for human leukocyte 5-lipoxygenase. Two clonal cell lines derived from the transfected cells expressed the enzymatic activity. When incubated with arachidonic acid (100 microM), the major 5-lipoxygenase products of 10,000 X g supernatants from these cells were 5-hydroxyeicosatetraenoic acid (5-HETE), and the nonenzymatic hydrolysis products of leukotriene (LT)A4. The ratio of 5-HETE to LT (between 6:1 and 9:1) was similar to that observed in leukocyte supernatants. Furthermore, incubation of 10,000 X g supernatants from the transfected cells with 5-hydroperoxyeicosatetraenoic acid (5-HPETE) (75 microM) resulted in the synthesis of LTA4 hydrolysis products. Control osteosarcoma cell supernatants produced no 5-HETE or LT from arachidonic acid or 5-HPETE. Maximal activity of the expressed enzyme required Ca2+, ATP, and two cellular stimulatory factors prepared from human leukocytes. Immunoblot analysis of supernatants from the osteosarcoma cell clones revealed an immunoreactive 80,000-dalton band that was indistinguishable from the band observed in leukocyte supernatants. Therefore, the expressed enzyme was functional and exhibited characteristics that were identical to those of human leukocyte 5-lipoxygenase. When intact transfected osteosarcoma cells were challenged with ionophore A 23187, no 5-lipoxygenase products were formed. If arachidonic acid was added along with the ionophore, the cells synthesized 5-HETE and the nonenzymatic hydrolysis products of LTA4. These results verify that the cDNA used to transfect the osteosarcoma cells encodes for 5-lipoxygenase. Furthermore, these studies offer independent evidence that this single protein possesses both 5-lipoxygenase and LTA4 synthase activity, as has been reported previously from enzyme purification data.

Regulation of the human leukocyte 5-lipoxygenase: stimulation by micromolar Ca 2+ levels and phosphatidylcholine vesicles

Human leukocyte 5-lipoxygenase (EC 1.13.11.12) is unique among the human lipoxygenases not only in its requirement for free ionized calcium, but also in its regulation by a membrane-associated stimulatory factor, the 100000 × g pellet. In the present study, phosphatidylcholine (PC) vesicles, in the absence of 100000 × g pellet, exhibited a dose-dependent stimulatory activity on the 5-lipoxygenase, which was at least as effective as the 100000 × g pellet. Furthermore, the enzyme was activated by isolated human neutrophil plasma membranes and to a lesser degree by endoplasmic reticulum. The chemoattractant peptide fMet-Leu-Phe (0.1 μM), GTP (10 μM), toxin from bacterium Bordetella pertussis (islet activating protein, 5 μg/ml) and their various combinations were unable to modulate the enzymatic activity of the 5-lipoxygenase. Stimulation of the 5-lipoxygenase by relatively low levels of free ionized calcium was observed both in the presence of the pellet and PC vesicles: maximal stimulation was seen at about 10 μM Ca 2+. The human leukocyte leukotriene A 4 synthase activity also exhibited a similar requirement for free calcium ions. The present study indicates that the membrane-associated stimulatory factor of the human leukocyte 5-lipoxygenase may be replaced by PC vesicles. Moreover, the 5-lipoxygenase and leukotriene A 4 synthase activities require significantly lower Ca 2+ levels for maximal activation than has been reported previously.

Reversible, calcium-dependent membrane association of human leukocyte 5-lipoxygenase.

Maximal activity of human leukocyte 5-lipoxygenase requires Ca2+, ATP, a microsomal membrane preparation, and two cytosolic stimulatory factors. We report here some effects of Ca2+ on the physical properties of the 5-lipoxygenase. When leukocytes were homogenized in the presence of 2 mM EDTA, 5-lipoxygenase was found to be a soluble enzyme. However, when Ca2+ was added to homogenization buffers at 0-1 mM in excess of EDTA, increasing quantities of the enzyme were recovered in the microsomal membrane fraction (100,000 X g pellet). The membrane-associated enzyme was resolubilized by washing pellet preparations in buffers containing 2 mM EDTA and was partially purified by anion-exchange chromatography. Studies of the stimulatory-factor requirements of the membrane-associated, resolubilized, and partially purified enzyme indicated that one of the cytosolic 5-lipoxygenase stimulatory factors exhibited a reversible, Ca2+-dependent membrane association, similar to that of the enzyme itself. Ca2+ also caused a destabilization of the 5-lipoxygenase. Homogenates prepared in the presence of Ca2+ contained lower total enzyme activity, and retention of activity in these samples over time was also diminished.

Demonstration of multiple regulatory factors for purified human leukocyte 5-lipoxygenase

The human leukocyte 5-lipoxygenase is a unique enzyme in its class because of its involvement in the synthesis of the biologically active leukotrienes. Furthermore, unlike most other lipoxygenases, this enzyme requires multiple stimulatory factors for maximal activity. These include Ca 2+, ATP, and three non-dialyzable cellular components, two cytosolic, and one membrane-associated. The mechanism of action of these factors is not yet well understood; however, a Ca 2+-dependent association of the enzyme and one of the cytosolic factors with the membrane has been demonstrated. These findings suggest that stimulation of the leukocyte, resulting in an increased intracellular Ca 2+ concentration, may result in the translocation of the enzyme and the factor to a membrane site, thereby facilitating the interaction of these two proteins with other enzymes involved in the 20:4 metabolic cascade. The development of a better understanding of these processes should not only help to define the biochemical basis for the regulation of leukotriene formation, but should also yield valuable information concerning the more general aspects of stimulus-response coupling in the leukocyte.