Regulation of lysophospholipase activity of the 85-kDa phospholipase A2 and activation in mouse peritoneal macrophages.
The regulation of the lysophospholipase activity of the 85-kDa cytosolic phospholipase A2 (PLA2) was studied in vitro and in stimulated macrophages. Bovine serum albumin was found to inhibit lysophospholipase activity of the recombinant 85-kDa PLA2 when assayed at a relatively low substrate concentration. Inhibition could be reversed if the substrate concentration was increased or if Ca2+ was present in the assay. Incubation of recombinant enzyme with macrophage membranes and lipid extracts from macrophage membranes resulted in the release of arachidonic acid, as well as, stearic acid, which is enriched at the sn-1 position of macrophage phospholipids. This suggests that with a bilayer substrate the PLA2 can sequentially deacylate the sn-2 then sn-1 acyl groups. This was verified by demonstrating that the phospholipids, phosphatidylcholine and phosphatidylinositol, were hydrolyzed to glycerophosphocholine and glycerophosphoinositol by incubation with recombinant 85-kDa PLA2. The 85-kDa enzyme was identified as the main lysophospholipase activity in mouse peritoneal macrophage cytosols. Addition of Ca2+ to the assay enhanced activity, but this effect decreased as the substrate concentration was increased. Incubation of macrophages with zymosan increased the lysophospholipase activity of the 85-kDa PLA2 in cytosols. Phosphorylation of recombinant PLA2 with mitogen-activated protein kinase resulted in an increase in lysophospholipase, as well as, PLA2 activity. In macrophages stimulated with zymosan release of stearic acid (18:0) and palmitic acid (16:0) was observed in addition to arachidonic acid (20:4). These results are consistent with a role of the 85-kDa PLA2 in regulating lysophospholipid levels in macrophages during zymosan stimulation.
de Carvalho
M G
MG
Department of Pediatrics, National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado 80206, USA.
Garritano
J
J
Leslie
C C
CC
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D003600:Cytosol / Q000201:enzymology; D004789:Enzyme Activation; D005227:Fatty Acids / Q000032:analysis / Q000378:metabolism; D005260:Female; D006706:Homeostasis; D007700:Kinetics; D008245:Lysophospholipase / Q000378:metabolism*; D008246:Lysophospholipids / Q000494:pharmacology; D017737:Macrophages, Peritoneal / Q000187:drug effects / Q000201:enzymology*; D008563:Membrane Lipids / Q000378:metabolism; D051379:Mice; D008813:Mice, Inbred ICR; D008970:Molecular Weight; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D011994:Recombinant Proteins / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000494:pharmacology; D014020:Tissue Extracts / Q000494:pharmacology; D015054:Zymosan / Q000494:pharmacology
D005227:Fatty Acids; D008246:Lysophospholipids; D008563:Membrane Lipids; D011994:Recombinant Proteins; D014020:Tissue Extracts; D012710:Serum Albumin, Bovine; D015054:Zymosan; D010741:Phospholipases A; D054467:Phospholipases A2; D008245:Lysophospholipase; D002118:Calcium