No abstract

5-Lipoxygenase (5-LO) catalysis is positively regulated by Ca2؉ ions and phospholipids that both act via the Nterminal C2-like domain of 5-LO. Previously, we have shown that 1-oleoyl-2-acetylglycerol (OAG) functions as an agonist for human polymorphonuclear leukocytes (PMNL) in stimulating 5-LO product formation. Here we have demonstrated that OAG directly stimulates 5-LO catalysis in vitro. In the absence of Ca 2؉ (chelated using EDTA), OAG strongly and concentration-dependently stimulated crude 5-LO in 100,000 ؋ g supernatants as well as purified 5-LO enzyme from PMNL. Also, the monoglyceride 1-O-oleyl-rac-glycerol and 1,2-dioctanoyl-sn-glycerol were effective, whereas various phospholipids did not stimulate 5-LO. However, in the presence of Ca In the biosynthesis of leukotrienes (LTs) 1 , 5-lipoxygenase (5-LO) catalyzes the initial oxygenation of arachidonic acid (AA) leading to 5-HPETE, which is further metabolized by 5-LO to LTA 4 (for review, see Refs. 1 and 2). The mechanisms leading to activation of 5-LO in the cell are complex, and the enzymatic activity of 5-LO is tightly controlled. At its active site, 5-LO contains an essential nonheme-bound iron (3), which, in the resting state, is in the ferrous (Fe 2ϩ ) form and requires oxidation to the ferric (Fe 3ϩ ) state to enter the catalytic cycle (4, 5).In the absence of stimulating co-factors, 5-LO activity in vitro is low, and it has been found that Ca 2ϩ , ATP, and phospholipids are required for full enzyme activity (for review, see Refs. 1 and 2). Also, a threshold level of lipid hydroperoxides (LOOH) is necessary for initial enzyme activation, converting ferrous to ferric iron (4, 6, 7). For cellular 5-LO activation, elevated Ca 2ϩ levels (8), phosphorylation events by tyrosine kinases (9) and by members of the mitogen-activated protein kinase family (10, 11), an elevated peroxide tone (12-14), and nuclear membrane association (15, 16), including co-localization with the 5-LO-activating protein (17, 18), are determinants. Nevertheless, the precise pathway(s) and regulatory mechanisms of 5-LO activation in the cell are incompletely understood.Based on theoretical models of the tertiary structure, 5-LO consists of a catalytic and an N-terminal C2-like ␤-barrel domain (19 -22). The C2-like domain binds Ca 2ϩ and phosphatidylcholine (PC) (19, 21) and targets 5-LO to the nuclear membrane (20,21). Ca 2ϩ and/or phospholipids can strongly augment 5-LO activity in vitro but also may have no stimulatory effect, which depends on the assay conditions (see Refs. 1 and 23 and references therein). Ca 2ϩ has been shown to increase the affinity of 5-LO toward AA (24), to facilitate membrane association and binding to PC vesicles (21, 25), and appears to reduce the requirement of 5-LO for activating lipid hydroperoxides (26). For stimulation by phospholipids, apparently only the zwitterionic PC and a non-physiological cationic phospholipid (but not phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, or diacylglycerol (DAG)) stimulate 5-LO reactions (27,...

Mammalian ferritin subunit synthesis is controlled at the translational level by the iron regulatory protein 1 (IRP1)/iron responsive element (IRE) interaction. Insect haemolymph ferritin subunit messages have an IRE in the 5'-untranslated region (UTR). We have shown that recombinant M. sexta IRP1 represses the in vitro translation of both the heavy and light chain ferritin subunits from this species without altering transcription. Deletion of either the 5'-UTR or the IRE from the mRNA abolishes IRP1 repression. Our studies indicated that the translational control of ferritin synthesis by IRP/IRE interaction could occur in insects in a manner similar to that of mammals. To our knowledge, this is the first report of the control of insect ferritin synthesis by IRP1/IRE interaction. Furthermore, this is the first indication that the synthesis of a secreted ferritin subunit can also be controlled in this manner.

No abstract

Diacylglycerides (DAGs) such as 1-oleoyl-2-acetyl-sn-glycerol (OAG) stimulate 5-lipoxygenase (5-LO) enzyme activity and function as agonists for human polymorphonuclear leukocytes (PMNL) to induce 5-LO product synthesis. Here, we addressed the role of endogenous DAG generation in agonist-induced 5-LO activation in human PMNL. Preincubation of PMNL with the phospholipase D (PLD) inhibitor 1-butanol potently suppressed 5-LO product synthesis induced by the Ca(2)(+) ionophore A23187 or thapsigargin (TG) and blocked A23187-evoked translocation of 5-LO from the cytosol to the nuclear membrane, analyzed by subcellular fractionation as well as by indirect immunofluorescence microscopy. Tertiary-butanol, a rather poor inhibitor of PLD, caused only moderate suppression of 5-LO and hardly inhibited 5-LO translocation. Interestingly, 1-butanol failed to inhibit 5-LO product formation when PMNL were stimulated with OAG (30 microM). Moreover, coincubation of A23187- or TG-stimulated PMNL with OAG reversed inhibition of 5-LO product formation by 1-butanol in a concentration-dependent manner (EC(50), approximately 1 muM) and also restored 5-LO translocation. In addition, inhibition of phosphatidic acid phosphatase (PA-P) by propranolol or bromoenol lactone caused suppression of 5-LO product formation and of translocation, which could be reversed by addition of exogenous OAG. Together, our data suggest that in agonist-stimulated PMNL, the endogenous formation of DAGs via the PLD/PA-P pathway determines 5-LO activation.