Abstract
Phospholipase A2 (PLA2), EC 3.1.1.4, which catalyzes the release of free fatty acids from the sn-2 position of glycerophospholipids, has been extensively studied from the viewpoint of eicosanoid production (Arita, H., Nakano, T., and Hanasaki, K. (1989) Prog. Lipid Res. 28, 273-301). Several lines of evidence suggest that extracellular PLA2 is pathophysiologically related to some disorders, including inflammation and hypersensitivity. Despite this, little is known of the precise mechanism of the pathological processes as well as their intrinsic correlation with dysfunction. Here, we report a novel PLA2 action on the proliferation of Swiss 3T3 fibroblasts via specific binding sites of approximately Mr 200,000. Pancreatic type PLA2 in the active form specifically recognized the sites and stimulated thymidine incorporation in DNA. Its inactive zymogen and other PLA2s from platelets, snake, and bee venoms showed much lesser activities. Although the physiological significance remains to be identified, our finding is the first to offer a new viewpoint on the effect of mammalian extracellular PLA2 on cellular function.
Highlights
Cells via Specific BindingSite*Distinctive roles for this type of enzyme. With theuse of Swiss 3T3 fibroblast cells and PLA2-I from several animal species, we have obtained evidence for the new type of function of PLA2-I,possibly unrelated to itsphospholipid-hydrolyzing properties
Materials-Porcine Phospholipase Az (PLAz)-Iwas purified to homogeneity according to themethod of Puijk et al [16]
Phospholipase Az (PLAz),EC 3.1.1.4, whichcata- form were purified from pancreatic juice [17], and recombinant hulyzes thereleaseof free fatty acidsfromthe sn-2 man PLAz-I and its pro-form produced by Saccharomycescereuisiae position of glycerophospholipids, has beeenxtensively [18]were used
Summary
Distinctive roles for this type of enzyme. With theuse of Swiss 3T3 fibroblast cells and PLA2-I from several animal species, we have obtained evidence for the new type of function of PLA2-I,possibly unrelated to itsphospholipid-hydrolyzing properties. Binding Experiments-Confluent 3T3 cells grown in 35-mm diameter dishes were washed twice with phosphate-buffered saline and incubated with various concentrations of lZ5I-PLA2-iIn 1 ml of matory processes [1, 2]. The specific binding is defined by subtracting the nonspecific binding, the amount of '251-PLAz-bI ound in the simultaneous addition of the unlabeled porcine pancreatic PLA, (500 nM), from the total binding. Competition experiments on 3T3 cells were performed by incubating with 1nM 'zsII-PLA2-iIn the presence of rat via enhancement of gene transcription [5,6,7,8,9]. 150-mm diameter dishes (1.2 X lo cells) were incubated with 2 nM '251-PLA,-I(porcine) in the absence or presence of 500 nM unlabeled porcine PLAz-I for 3 hat 4 "C.
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