Ca(2+)-independent cytosolic phospholipase A2 from macrophage-like P388D1 cells. Isolation and characterization.
A novel form of an ATP-regulated, oligomeric, Ca(2+)-independent phospholipase A2 (iPLA2) has been purified from the cytosol of the murine macrophage-like cell line P388D1. The purification procedure included ammonium sulfate precipitation and sequential column chromatography on octyl-Sepharose, ATP-agarose, Mono Q fast protein liquid chromatography (FPLC), and hydroxyapatite FPLC. The resulting enzyme preparation was purified over 400,000-fold with a final specific activity of approximately 5 mumol/min/mg using a mixed micelle assay system of Triton X-100 and dipalmitoyl phosphatidylcholine (PC). The purified enzyme was Ca(2+)-independent and did not show a preference for either sn-2 arachidonic acid or sn-1 alkyl-ether containing phospholipids when utilizing mixed micelles as substrate. It was found to hydrolyze dipalmitoyl-PC approximately 4-fold faster than 1-palmitoyl-2-arachidonyl-PC and approximately 15-fold faster than 1-O-hexadecyl-2-arachidonyl-PC. Triton X-100 increased the P388D1 iPLA2 activity with optimal activity found at a Triton/phospholipid molar ratio of 4:1. The purified enzyme was activated 2-6-fold by ATP as well as other di- and triphosphate nucleosides. This activation was sensitive to the concentration of Triton X-100 present in the assay. SDS-polyacrylamide gel electrophoresis carried out on the purified enzyme yielded a single major band at a molecular weight of about 80,000. However, radiation inactivation experiments, carried out on the cell homogenate, demonstrated a target size of 337 +/- 25 kDa, indicating that the catalytically active iPLA2 exists as a large oligomeric complex, either through self-aggregation or association of the enzyme with other proteins.
Ackermann
E J
EJ
Department of Chemistry, University of California, San Diego, La Jolla 92093-0601.
Kempner
E S
ES
Dennis
E A
EA
D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D002118:Calcium / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology; D004789:Enzyme Activation; D007941:Leukemia P388 / Q000201:enzymology*; D008264:Macrophages / Q000201:enzymology*; D008861:Microsomes / Q000201:enzymology; D008970:Molecular Weight; D017830:Octoxynol / Q000737:chemistry; D010741:Phospholipases A / Q000302:isolation & purification / Q000378:metabolism*; D054467:Phospholipases A2; D013379:Substrate Specificity
D000255:Adenosine Triphosphate; D017830:Octoxynol; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium