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.

The extracellular phospholipase A2s (PLA2) from cobra venom, rattlesnake venom, and porcine pancreas were analyzed by radiation inactivation to determine their functional aggregation states. The analysis was performed in the presence of the protein transferrin at two different concentrations of PLA2: 5 micrograms/ml. The small size of these proteins necessitated the use of high radiation dosages. The catalytic activity of all samples decreased as a single exponential as a function of radiation dosage, to > 97% inactivation. Target size analysis of these curves yielded sizes corresponding to dimers for all three PLA2s, indicating that all three enzymes exist as dimers or larger aggregates under the conditions studied. An analysis of the amount of intact protein remaining by sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed that the loss of protein also followed a dimeric size for all three PLA2s. The loss of protein as a dimer indicates that transfer of radiation energy is occurring between polypeptides.