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Regulation of lysophospholipase activity of the 85-kDa phospholipase A2 and activation in mouse peritoneal
M G de Carvalho1, J Garritano, C C Leslie
1Department of Pediatrics, National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado 80206, USA.
Abstract:
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.
Insights
The 85-kDa cytosolic phospholipase A2 (PLA2) enzyme shows significant lysophospholipase activity, crucial for regulating lysophospholipid levels in macrophages. Its activity is modulated by substrate concentration, calcium ions, and phosphorylation.
Area of Science:
- Biochemistry
- Cell Biology
- Enzymology
Background:
- The 85-kDa cytosolic phospholipase A2 (PLA2) plays a role in cellular lipid metabolism.
- Understanding the regulation of its lysophospholipase activity is important for cellular signaling and lipid homeostasis.
Purpose of the Study:
- To investigate the regulation of the 85-kDa cytosolic phospholipase A2 (PLA2) lysophospholipase activity.
- To determine the enzyme's role in macrophage lipid metabolism, particularly during stimulation.
Main Methods:
- In vitro assays using recombinant 85-kDa PLA2 with varying substrate concentrations and calcium.
- Incubation of the enzyme with macrophage membranes and lipid extracts.
- Analysis of lipid products released from phospholipids.
- Assessing enzyme activity in stimulated mouse peritoneal macrophages.
Main Results:
- Bovine serum albumin inhibited lysophospholipase activity, reversible by increased substrate or Ca2+.
- The 85-kDa PLA2 sequentially deacylated sn-2 then sn-1 acyl groups of phospholipids.
- The enzyme was identified as the primary lysophospholipase in macrophage cytosols.
- Zymosan stimulation increased lysophospholipase activity, accompanied by the release of stearic and palmitic acids.
- Mitogen-activated protein kinase phosphorylation enhanced both PLA2 and lysophospholipase activities.
Conclusions:
- The 85-kDa PLA2 functions as a key lysophospholipase in macrophages.
- Enzyme activity is regulated by substrate availability, Ca2+, and phosphorylation.
- The enzyme contributes to regulating lysophospholipid levels in macrophages during inflammatory responses.