The 5-lipoxygenase pathway in arterial wall biology and atherosclerosis

Leukotrienes (LTs) are powerful inflammatory lipid mediators derived from the 5-lipoxygenase (5-LO) cascade of arachidonic acid. Recent clinical, population genetic, cell biological, and mouse studies indicate participation of the 5-LO pathway in atherogenesis and arterial wall remodeling. 5-LO is expressed by leukocytes including blood monocytes, tissue macrophages, dendritic cells, neutrophils, and mast cells. LTB4 and the cysteinyl LTs LTC4, LTD4, and LTE4, act through two BLT and two cysLT receptors that are differentially expressed on hematopoietic and arterial wall cells. The precise roles of LTs or the LT receptors in cardiovascular physiology remain largely to be explored. In this review, we will discuss what is currently known about the 5-LO atherosclerosis connection. We will attempt to propose strategies to further explore potential links between the 5-LO pathway and blood vessel physiology and disease progression.     

Protein kinase A inhibits leukotriene synthesis by phosphorylation of 5-lipoxygenase on serine 523

Leukotrienes (LTs) are lipid messengers generated by leukocytes that drive inflammation and modulate neighboring cell function. The synthesis of LTs from arachidonic acid is initiated by the enzyme 5-lipoxygenase (5-LO). We report for the first time that LT synthesis is inhibited by the direct action of protein kinase A (PKA) on 5-LO. The catalytic subunit of PKA directly phosphorylated 5-LO in vivo and in vitro and inhibited activity in intact cells and in vitro. Mutation of Ser-523 on human 5-LO prevented phosphorylation by PKA and restored LT synthesis. Treatment with PKA activators inhibited LTB(4) synthesis in 3T3 cells expressing wild type 5-LO but not in cells expressing the S523A mutant of 5-LO. The mechanism of inhibition of LTB(4) synthesis did not involve either reduced membrane association of activated 5-LO or redistribution of 5-LO from the nucleus to the cytoplasm. Instead, PKA phosphorylation of recombinant 5-LO inhibited in vitro activity, as did co-transfection of cells with 5-LO plus the catalytic subunit of PKA. Also, substitution of Ser-523 with glutamic acid, mimicking phosphorylation, resulted in the total loss of 5-LO activity. These results indicate that PKA phosphorylates 5-LO on Ser-523, which inhibits the catalytic activity of 5-LO and reduces cellular LT generation. Thus, PKA activation, as can occur in response to adenosine, prostaglandin E(2), beta-adrenergic agonists, and other mediators, is a means of directly reducing 5-LO activity and LT synthesis that may be important in limiting inflammation and maintaining homeostasis.     

Suppression of 5-lipoxygenase activity by anionic cholesterol derivatives

5-Lipoxygenase (5-LO) is a key enzyme involved into biosynthesis of leukotrienes (LTs), mediating the host defense system, and acting simultaneously as inflammatory agents. In this work the effect of anionic cholesterol derivatives on 5-LO activity has been investigated. Cholesterol sulfate activates human polymorphonuclear leukocytes (PMNL) and stimulates their adhesion to endothelium and collagen. Cholesterol sulfate and cholesterol phosphate suppressed leukotriene production in PMNL and in rat basophil leukemia (RBL-1) cell line as well as in homogenates of these cells. Kinetic characteristics of the effect of anionic cholesterol derivatives on leukotriene synthesis have been obtained. In all experiments cholesterol phosphate (charge-2) was shown to be more potent inhibitor than cholesterol sulfate (charge-1). We believe that this fact highlights the importance of negatively charged ester groups for suppression of 5-LO activity.     

Cholesterol and its anionic derivatives inhibit 5-lipoxygenase activation in polymorphonuclear leukocytes and MonoMac6 cells

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.     

5-Lipoxagenase deficiency attenuates L-NAME-induced hypertension and vascular remodeling

BackgroundAbnormalities of the L-arginine-nitric oxide pathway induce hypertension. 5-Lipoxygenase (5-LO) is the key enzyme involved in synthesis of leukotrienes (LTs). However, whether nitricoxide synthase dysfunction induces hypertensive vascular remodeling by regulating 5-LO activity and its downstream inflammatory metabolites remains unknown.Methods and resultsSix-week L-NAME treatment significantly induced hypertension and vascular remodeling in both wild-type (WT) and 5-LO–knockout (5-LO–KO) mice, and blood pressure in caudal and carotid arteries was lower in 5-LO–KO than WT mice with L-NAME exposure. On histology, L-NAME induced less media thickness, media-to-lumen ratio, and collagen deposition and fewer Ki-67–positive vascular smooth muscle cells (VSMCs) but more elastin expression in thoracic and mesenteric aortas of 5-LO–KO than L-NAME–treated WT mice. L-NAME significantly increased LT content, including LTB4 and cysteinyl LT (CysLTs), in plasma and neutrophil culture supernatants from WT mice. On immunohistochemistry, L-NAME promoted the colocalization of 5-LO and 5-LO–activating protein on the nuclear envelope of cultured neutrophils, which was accompanied by elevated LT content in culture supernatants. In addition, LTs significantly promoted BrdU incorporation, migration and phenotypic modulation in VSMCs.ConclusionL-NAME may activate the 5-LO/LT pathway in immune cells, such as neutrophils, and promote the products of 5-LO metabolites, including LTB4 and CysLTs, which aggravate vascular remodeling in hypertension. 5-LO deficiency may protect against hypertension and vascular remodeling by reducing levels of 5-LO downstream inflammatory metabolites.     

Inhibition of 5-lipoxygenase activity in mice during cuprizone-induced demyelination attenuates neuroinflammation, motor dysfunction and axonal damage

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS). Increased expression of 5-lipoxygenase (5-LO), a key enzyme in the biosynthesis of leukotrienes (LTs), has been reported in MS lesions and LT levels are elevated in the cerebrospinal fluid of MS patients. To determine whether pharmacological inhibition of 5-LO attenuates demyelination, MK886, a 5-LO inhibitor, was given to mice fed with cuprizone. Gene and protein expression of 5-LO were increased at the peak of cuprizone-induced demyelination. Although MK886 did not attenuate cuprizone-induced demyelination in the corpus callosum or in the cortex, it attenuated cuprizone-induced axonal damage and motor deficits and reduced microglial activation and IL-6 production. These data suggest that during cuprizone-induced demyelination, the 5-LO pathway contributes to microglial activation and neuroinflammation and to axonal damage resulting in motor dysfunction. Thus, 5-LO inhibition may be a useful therapeutic treatment in demyelinating diseases of the CNS.     

Deficiency of 5-lipoxygenase accelerates renal allograft rejection in mice.

Acute renal allograft rejection is associated with alterations in renal arachidonic acid metabolism, including enhanced synthesis of leukotrienes (LTs). LTs, the products of the 5-lipoxygenase (5-LO) pathway, are potent lipid mediators with a broad range of biologic activities. Previous studies, using pharmacological agents to inhibit LT synthesis or activity, have implicated these eicosanoids in transplant rejection. To further investigate the role of LTs in acute graft rejection, we transplanted kidneys from CByD2F1 mice into fully allogeneic 129 mice that carry a targeted mutation in the 5lo gene. Unexpectedly, allograft rejection was significantly accelerated in 5-LO-deficient mice compared with wild-type animals. Despite the marked reduction in graft survival, the 5lo mutation had no effect on the hemodynamics or morphology of the allografts. Although LTB4 levels were reduced, renal thromboxane B2 production and cytokine expression were not altered in 5-LO-deficient allograft recipients. These findings suggest that, along with their proinflammatory actions, metabolites of 5-LO can act to enhance allograft survival.     

Characterization and cellular localization of human 5-lipoxygenase and its protein isoforms 5-LOΔ13, 5-LOΔ4 and 5-LOp12

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.     

Caffeic acid phenethyl ester and its amide analogue are potent inhibitors of leukotriene biosynthesis in human polymorphonuclear leukocytes

Background

5-lipoxygenase (5-LO) catalyses the transformation of arachidonic acid (AA) into leukotrienes (LTs), which are important lipid mediators of inflammation. LTs have been directly implicated in inflammatory diseases like asthma, atherosclerosis and rheumatoid arthritis; therefore inhibition of LT biosynthesis is a strategy for the treatment of these chronic diseases.

Methodology/Principal Findings

Analogues of caffeic acid, including the naturally-occurring caffeic acid phenethyl ester (CAPE), were synthesized and evaluated for their capacity to inhibit 5-LO and LTs biosynthesis in human polymorphonuclear leukocytes (PMNL) and whole blood. Anti-free radical and anti-oxidant activities of the compounds were also measured. Caffeic acid did not inhibit 5-LO activity or LT biosynthesis at concentrations up to 10 µM. CAPE inhibited 5-LO activity (IC₅₀ 0.13 µM, 95% CI 0.08–0.23 µM) more effectively than the clinically-approved 5-LO inhibitor zileuton (IC₅₀ 3.5 µM, 95% CI 2.3–5.4 µM). CAPE was also more effective than zileuton for the inhibition of LT biosynthesis in PMNL but the compounds were equipotent in whole blood. The activity of the amide analogue of CAPE was similar to that of zileuton. Inhibition of LT biosynthesis by CAPE was the result of the inhibition of 5-LO and of AA release. Caffeic acid, CAPE and its amide analog were free radical scavengers and antioxidants with IC₅₀ values in the low µM range; however, the phenethyl moiety of CAPE was required for effective inhibition of 5-LO and LT biosynthesis.

Conclusions

CAPE is a potent LT biosynthesis inhibitor that blocks 5-LO activity and AA release. The CAPE structure can be used as a framework for the rational design of stable and potent inhibitors of LT biosynthesis.     

The nuclear membrane leukotriene synthetic complex is a signal integrator and transducer

Leukotrienes (LTs) are lipid-signaling molecules derived from arachidonic acid (AA) that initiate and amplify inflammation. To initiate LT formation, the 5-lipoxygenase (5-LO) enzyme translocates to nuclear membranes, where it associates with its scaffold protein, 5-lipoxygenase–activating protein (FLAP), to form the core of the multiprotein LT synthetic complex. FLAP is considered to function by binding free AA and facilitating its use as a substrate by 5-LO to form the initial LT, LTA₄. We used a combination of fluorescence lifetime imaging microscopy, cell biology, and biochemistry to identify discrete AA-dependent and AA-independent steps that occur on nuclear membranes to control the assembly of the LT synthetic complex in polymorphonuclear leukocytes. The association of AA with FLAP changes the configuration of the scaffold protein, enhances recruitment of membrane-associated 5-LO to form complexes with FLAP, and controls the closeness of this association. Granulocyte monocyte colony–stimulating factor provides a second AA-independent signal that controls the closeness of 5-LO and FLAP within complexes but not the number of complexes that are assembled. Our results demonstrate that the LT synthetic complex is a signal integrator that transduces extracellular signals to modulate the interaction of 5-LO and FLAP.     

5-脂氧化酶与动脉粥样硬化的研究进展

动脉粥样硬化（AS）及其并发症是当今世界上导致死亡的最常见的病因之一。AS发病机制有多种学说,其中动脉粥样硬化是一种慢性炎症性疾病的学说已为广大学者所接受。白三烯（LTs）是促进炎症反应的脂类调控因子,在心血管组织中具有强烈的致炎作用,5-脂氧化酶（5-LO）能够氧化花生四烯酸生成LTs等花生酸类物质。近年对动脉粥样硬化的研究中发现5-LO途径及其下游产物LTs对AS的形成、发展及动脉粥样硬化斑块的不稳定性有重要作用。本文结合国内外文献,对5-LO基因结构、蛋白结构和代谢活性等生物学特性以及5-LO及其下游产物与动脉粥样硬化发生、发展的关系进行了总结,对了解和研究动脉粥样硬化疾病及其治疗具有重要意义。     

Eugenol--the active principle from cloves inhibits 5-lipoxygenase activity and leukotriene-C4 in human PMNL cells

Polymorphonuclear leukocytes (PMNL) play an important role in the modulation of inflammatory conditions in humans. PMNL cells recruited at the site of inflammation, release inflammatory mediators such as leukotrienes, proteolytic enzymes and reactive oxygen species. Among these, leukotrienes are implicated in pathophysiology of allergic and inflammatory disorders like asthma, allergic rhinitis, arthritis, inflammatory bowel disease and psoriasis. 5-lipoxygenase (5-LO) is the key enzyme in biosynthetic pathway of leukotrienes. Our earlier studies showed that spice phenolic active principles significantly inhibit 5-LO enzyme in human PMNLs. In this study we have further characterized the inhibitory mechanism of eugenol, the active principle of spice-clove on 5-LO enzyme and also its effect on leukotriene C((4)) (LTC(4)). Substrate dependent enzyme kinetics showed that the inhibitory effect of eugenol on 5-LO was of a non-competitive nature. Further, eugenol was found to significantly inhibit the formation of LTC(4) in calcium ionophore A23187 and arachidonic acid (AA) stimulated PMNL cells. These data clearly suggest that eugenol inhibits 5-LO by non-competitive mechanism and also inhibits formation of LTC(4) in human PMNL cells and thus may have beneficial role in modulating 5-LO pathway in human PMNL cells.     

Expression of 5-lipoxygenase in specialized epithelial cells of nasopharyngeal-associated lymphoid tissue

Leukotrienes are lipid mediators that are produced primarily by certain types of leukocytes. The synthesis of the leukotriene LTB₄ is initiated by the enzyme 5-lipoxygenase and completed by LTA₄ hydrolase. Epithelial cells constitutively express LTA₄ hydrolase but normally lack 5-lipoxygenase. In this study, we report that the stratified squamous epithelial cells from inflamed or hyperplastic tissues of palatine and pharyngeal tonsils (nasopharyngeal-associated lymphoid tissue) express 5-lipoxygenase protein. The localization of 5-lipoxygenase was indicated by immunohistochemical staining and presence confirmed by immunoblot. Positive staining for 5-lipoxygenase in infiltrating leukocytes in inflamed tissues served as internal positive controls for immunohistochemical staining. Staining for 5-lipoxygenase in appendix tissue was negative for epithelial cells while positive for polymorphonuclear leukocytes, indicating that 5-lipoxygenase expression is not a general feature of epithelial cells in mucosa-associated lymphoid tissue. In tonsils, 5-lipoxygenase staining was pronounced in broad regions but reduced or absent in others, suggesting regional regulation of expression. Epithelial cells of tonsils were also positive for 5-lipoxygenase activating protein and leukotriene A₄ hydrolase, indicating a capacity to produce LTB₄. Taken together, these results suggest that the specialized epithelial cells of the mucosa-associated lymphoid tissue of human tonsils can synthesize LTB₄. This lipid mediator may serve to modulate the function of cells within the lymphoid tissue as well as promote an inflammatory response.     

FcepsilonRI- and Fcgamma receptor-mediated production of reactive oxygen species by mast cells is lipoxygenase- and cyclooxygenase-dependent and NADPH oxidase-independent

We investigated the enzymes responsible for FcepsilonRI-dependent production of reactive oxygen species (ROS) and the influence of ROS on mast cell secretory responses. 5-Lipoxygenase (5-LO) was the primary enzyme involved in ROS production by human mast cells (huMC) and mouse bone marrow-derived mast cells (mBMMC) following FcepsilonRI aggregation because incubation with 5-LO inhibitors (AA861, nordihydroguaiaretic acid, zileuton) but not a flavoenzyme inhibitor (diphenyleneiodonium) completely abrogated Ag-induced dichlorodihydrofluorescein (DCF) fluorescence. Furthermore, 5-LO-deficient mBMMC had greatly reduced FcepsilonRI-dependent DCF fluorescence compared with wild type mBMMC or those lacking a functional NADPH oxidase (i.e., gp91(phox)- or p47(phox)-deficient cells). A minor role for cyclooxygenase (COX)-1 in FcepsilonRI-dependent ROS production was demonstrated by inhibition of Ag-mediated DCF fluorescence by a COX-1 inhibitor (FR122047) and reduced DCF fluorescence in COX-1-deficient mBMMC. Complete abrogation of FcepsilonRI-dependent ROS production in mast cells had no effect on degranulation or cytokine secretion. In response to the NADPH oxidase-stimulating agents including PMA, mBMMC and huMC produced negligible ROS. IgG-coated latex beads did stimulate ROS production in huMC, and in this experiment 5-LO and COX again appeared to be the enzymatic sources of ROS. In contrast, IgG-coated latex bead-induced ROS production in human polymorphonuclear leukocytes occurred by the NADPH oxidase pathway. Thus mBMMC and huMC generate ROS by 5-LO and COX-1 in response to FcepsilonRI aggregation; huMC generate ROS upon exposure to IgG-coated latex beads by 5-LO and COX; and ROS appear to have no significant role in FcepsilonRI-dependent degranulation and cytokine production.     

Interplay of cysteinyl leukotrienes and TGF-β in the activation of hepatic stellate cells from Schistosoma mansoni granulomas

Hepatic stellate cells (HSCs) have a critical role in liver physiology, and in the pathogenesis of liver inflammation and fibrosis. Here, we investigated the interplay between leukotrienes (LT) and TGF-β in the activation mechanisms of HSCs from schistosomal granulomas (GR-HSCs). First, we demonstrated that GR-HSCs express 5-lipoxygenase (5-LO), as detected by immunolocalization in whole cells and confirmed in cell lysates through western blotting and by mRNA expression through RT-PCR. Moreover, mRNA expression of 5-LO activating protein (FLAP) and LTC₄-synthase was also documented, indicating that GR-HSCs have the molecular machinery required for LT synthesis. Morphological analysis of osmium and Oil-Red O-stained HSC revealed large numbers of small lipid droplets (also known as lipid bodies). We observed co-localization of lipid droplet protein marker (ADRP) and 5-LO by immunofluorescence microscopy. We demonstrated that GR-HSCs were able to spontaneously release cysteinyl-LTs (CysLTs), but not LTB_4, into culture supernatants. CysLT production was highly enhanced after TGF-β-stimulation. Moreover, the 5-LO inhibitor zileuton and 5-LO gene deletion were able to inhibit the TGF-β-stimulated proliferation of GR-HSCs, suggesting a role for LTs in HSC activation. Here, we extend the immunoregulatory function of HSC by demonstrating that HSC from liver granulomas of schistosome-infected mouse are able to release Cys-LTs in a TGF-β-regulated manner, potentially impacting pathogenesis and liver fibrosis in schistosomiasis.     

Five-lipoxygenase inhibitors can mediate apoptosis in human breast cancer cell lines through complex eicosanoid interactions.

Many arachidonic acid metabolites function in growth signaling for epithelial cells, and we previously reported the expression of the major arachidonic acid enzymes in human breast cancer cell lines. To evaluate the role of the 5-lipoxygenase (5-LO) pathway on breast cancer growth regulation, we exposed cells to insulinlike growth factor-1 or transferrin, which increased the levels of the 5-LO metabolite, 5(S)-hydrooxyeicosa-6E,8C,11Z,14Z-tetraenoic acid (5-HETE), by radioimmunoassay and high-performance liquid chromatography. Addition of 5-HETE to breast cancer cells resulted in growth stimulation, whereas selective biochemical inhibitors of 5-LO reduced the levels of 5-HETE and related metabolites. Application of 5-LO or 5-LO activating protein-directed inhibitors, but not a cyclooxygenase inhibitor, reduced growth, increased apoptosis, down-regulated bcl-2, up-regulated bax, and increased G1 arrest. Exposure of breast cancer cells to a 5-LO inhibitor up-regulated peroxisome proliferator-activated receptor (PPAR)a and PPARg expression, and these same cells were growth inhibited when exposed to relevant PPAR agonists. These results suggest that disruption of the 5-LO signaling pathway mediates growth arrest and apoptosis in breast cancer cells. Additional experiments suggest that this involves the interplay of several factors, including the loss of growth stimulation by 5-LO products, the induction of PPARg, and the potential activation of PPARg by interactions with shunted endoperoxides.     

Arachidonic acid promotes phosphorylation of 5-lipoxygenase at Ser-271 by MAPK-activated protein kinase 2 (MK2)

We demonstrated previously that 5-lipoxygenase (5-LO), a key enzyme in leukotriene biosynthesis, can be phosphorylated by p38 MAPK-regulated MAPKAP kinases (MKs). Here we show that mutation of Ser-271 to Ala in 5-LO abolished MK2 catalyzed phosphorylation and clearly reduced phosphorylation by kinases prepared from stimulated polymorphonuclear leukocytes and Mono Mac 6 cells. Compared with heat shock protein 27 (Hsp-27), 5-LO was a weak substrate for MK2. However, the addition of unsaturated fatty acids (i.e. arachidonate 1-50 microm) up-regulated phosphorylation of 5-LO, but not of Hsp-27, by active MK2 in vitro, resulting in a similar phosphorylation as for Hsp-27. 5-LO was phosphorylated also by other serine/threonine kinases recognizing the motif Arg-Xaa-Xaa-Ser (protein kinase A, Ca(2+)/calmodulin-dependent kinase II), but these activities were not increased by fatty acids. HeLa cells expressing wild type 5-LO or S271A-5-LO, showed prominent 5-LO activity when incubated with Ca(2+)-ionophore plus arachidonate. However, when stimulated with only exogenous arachidonic acid, activity for the S271A mutant was significantly lower as compared with wild type 5-LO. It appears that phosphorylation at Ser-271 is more important for 5-LO activity induced by a stimulus that does not prominently increase intracellular Ca(2+) and that arachidonic acid stimulates leukotriene biosynthesis also by promoting this MK2-catalyzed phosphorylation.     

Monocyte-derived soluble protein confers 5-lipoxygenase activity Ca2+-dependent

5-Lipoxygenase (5-LO) is a Ca2+-stimulated enzyme that initializes the formation of proinflammatory leukotrienes from arachidonic acid (AA). In this report, we demonstrate that a soluble protein of the monocytic cell line Mono Mac 6 confers 5-LO activity Ca2+-dependent in vitro. Thus, in broken cell preparations of human polymorphonuclear leukocytes (PMNL) and rat basophilic leukemia (RBL)-1 cells, 5-LO converted AA (>20 microM) in the absence of Ca2+, whereas Ca2+ was absolutely required for 5-LO activity in broken cell preparations of MM6 cells. 5-LO partially purified from MM6 cells was substantially active in the absence of Ca2+. Recombination experiments revealed that the cytosolic fraction of MM6 cells contains a factor that suppresses the activity of partially purified 5-LO from PMNL, RBL-1, and MM6 cells in the absence but not in the presence of Ca2+. Further characterization showed that this factor is a 80-100 kDa heat-sensitive protein.