Abstract
Studies are reported on the inhibition of phospholipase A2 (PLA2) from porcine pancreas, cobra (Naja naja) venom, and the P388D1 macrophage-like cell line by human recombinant lipocortin I and bovine lung calpactin I. Membrane vesicles prepared from 1-stearoyl,2-arachidonoyl phosphatidylcholine (PC) and other PCs were utilized as substrate. Binding studies using sucrose flotation gradients showed that both lipocortin I and calpactin I bind to these vesicles although less tightly than to vesicles prepared from anionic phospholipids or fatty acids. Binding to PC was somewhat enhanced by Ca2+. Inhibition of cobra venom PLA2 was not observed when PC vesicles were used as substrate but was when dipalmitoyl phosphatidylethanolamine was used. Both the pancreatic and macrophage enzymes were inhibited when acting on PC. Interestingly, the inhibition of the macrophage enzyme toward PC depended on the fatty acid attached to the sn-2 position of PC with arachidonate greater than oleate greater than palmitate. Inhibition was also highest at low [PC]; these inhibition results can be explained by the "substrate depletion model" (Davidson, F. F., Dennis, E. A., Powell, M., and Glenney, J. (1987) J. Biol. Chem. 262, 1698-1705). Experimental and theoretical considerations suggest that the in vitro inhibition by lipocortins of this macrophage PLA2 from a cell that makes lipocortin and is active in prostaglandin production is due to effects on substrate availability rather than direct inhibition.
Highlights
Title Binding and inhibition studies on lipocortins using phosphatidylcholine vesicles and phospholipase A2 from snake venom, pancreas, and a macrophage-like cell line
Binding studies using sucrose flotation gradients showed that both lipocortin I and calpactin I bind to these vesicles less tightly than to vesicles prepared from anionic phospholipids or fatty acids
Following our previous studies on “substrate depletion” [2], we have investigated the ability of human recombinant lipocortin I and bovine lung calpactin I to inhibit phospholipase A2 (PLA*)’ [3] from
Summary
THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc. Binding and Inhibition Studies on Lipocortins Using Phosphatidylcholine Vesicles and Phospholipase A2 from Snake Venom, Pancreas, and a Macrophage-like Cell Line*. Studies are reported on the inhibition of phospholipase AZ (PLA2) from porcine pancreas, cobra (Nuju naja) venom, and the P388D1 macrophage-like cell line by human recombinant lipocortin I and bovine lung calpactin I. PLA2 was not observed when PC vesicles were used as substrate but was when dipalmitoyl phosphatidylethanolamine was used Both the pancreatic and macrophage enzymes were inhibited when acting on PC. The mechanism of PLA, inhibition by these proteins in the only other commonly used in uitro assay system (phospholipid vesicles) warranted further study If it could be ascertained whether in vitro inhibition always involves depletion effects or sometimes works by a different mechanism perhaps more progress could be made toward quantitating the effect and determining the likely relevance of these proteins as inhibitors of PLA2s as discussed in a recent commentary [16]. A preliminary report has appeared [17]
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