Purification of a 110-kilodalton cytosolic phospholipase A2 from the human monocytic cell line U937.

Abstract

The major dithiothreitol-resistant phospholipase A2 activity present in the cytosol of U937 cells has been purified >200,000-fold by sequential chromatography on phenyl-SPW, heparin-Sepharose CL-6B, high-performance hydroxylapatite, TSK-gel G3000-SW, and Mono Q columns. This 110-kDa cytosolic phospholipase A2 is distinct from the relatively small (14-kDa) dithiothreitol-sensitive phospholipases A2 that are secreted from many cell types. This additional phospholipase A2 selectively hydrolyzes fatty acid at the sn-2 position of the glycerol and favors phospholipids containing arachidonic acid, which is the rate-limiting precursor for prostaglandin and leukotriene production. Interestingly, a >5-fold increase in phospholipase A2 activity is noted as the calcium concentration increases from the levels found in resting cells to those observed in activated macrophages. We suggest that this enzyme and not the previously described secretory phospholipase A2 is activated by cytosolic effectors such as GTP-binding regulatory proteins and protein kinases to initiate the production of prostaglandins, leukotrienes, and plateletactivating factor. To distinguish this cytosolic enzyme from the previously described secretory ones, we suggest referring to it as cPLA2 for cytosolic phospholipase A2 and collectively referring to the secretory phospholipases A2 as sPLA2s.Arachidonic acid is released from the sn-2 position of the glycerol in membrane phospholipids by an activated form of phospholipase A2 (PLA2) to initiate the synthesis of two potent classes ofinflammatory mediators, prostaglandins and leukotrienes (1-3). The other product of the PLA2 reaction is a lysophospholipid, which can serve as a precursor to the inflammatory mediator platelet-activating factor (PAF) (4). It is widely held that the production of arachidonic acid and lysophospholipid represents the rate-limiting step in the production of prostaglandins, leukotrienes, and plateletactivating factor. Therefore, agents that stimulate the production of these inflammatory mediators are likely to do so by modulating the activity of PLA2. However, the mechanism by which PLA2 activity is regulated remains poorly understood. To date, most of the direct studies on PLA2 have been structural or mechanistic and have focused on the extracellular enzymes isolated from snake venom or mammalian pancreas (5-9). Recently, the secretory granules of platelets and neutrophils have been shown to contain phospholipases A2 (PLA2s) that are structurally similar to the venom and pancreatic enzymes (10-12). There is accumulating evidence, however, that eicosanoid production is regulated by a PLA2 located in the cytosol.A number of different studies have shown that when cells are treated with phorbol 12-myristate 13-acetate (PMA), a potent activator of the cytosolic enzyme protein kinase C, an increase in prostaglandin, leukotriene, and platelet-activating factor production is observed (13-16). Interestingly, when the PLA2 activity present in the cytosolic fraction of PMAtreated cel...

Polychlorinated biphenyls induce arachidonic acid release in human platelets in a tamoxifen sensitive manner via activation of group IVA cytosolic phospholipase A2-α

“…2A). Pyrrolidine-1 was approximately 150 times more potent than AACOCF 3 and displayed an IC 50 value of 38 nM. BEL demonstrated a reduction of 12-HETE synthesis by 52% at the highest dose tested (30 μM).…”

“…Preincubation of human platelets for 10 min with increasing concentrations of pyrrolidine-1, AACOCF 3 or BEL led to decreased amounts of 12-HETE in a dose-dependent manner after stimulation with CB52 ( Fig. 2A).…”

“…Therefore, we investigated the involvement of PLA 2 enzymes in PCB-induced release of arachidonic acid in human platelets. To discriminate between secretory PLA 2 (sPLA 2 ) and intracellular PLA 2 -mediated arachidonic acid release, we employed the PLA2 inhibitors pyrrolidine-1, AACOCF 3 and BEL. Pyrrolidine-1 is a potent and selective inhibitor of cPLA 2 -α [48,49].…”

“…PLA 2 enzymes are a diverse family consisting of at least 16 different enzymes [2]. The intracellular group IV cytosolic PLA 2 -α (cPLA 2 ) [3,4] has attracted much interest in the biosynthesis of eicosanoids since it preferentially release arachidonic acid from membrane phospholipids. Interestingly, macrophages from mice deficient in cPLA 2 -α produced no prostaglandins or leukotrienes and various disease models have now been studied in these animals [5].…”

H2O2 renders cells accessible to lysis by exogenous phospholipase A2: a novel mechanism for cell damage in inflammatory processes

“…"IVe intracellular cytosolic PLA2 (cPLA2) preferentially hyd ~'olyses arachidonic acid (ArAc) containing PL, thus producil~g the precursor of the eicosanoids, which are mediators of cell activation in inflammatory processes [3]. The secreted PLAz (sPLAz) is found at high levels in body fluids in inflamn~atory diseases, such as septic shock, arthritis, acute lung il jury, psoriasis and bowel disease, and has been implicated il the pathophysiology of these diseases [4].…”