Skip to main content
eScholarship
Open Access Publications from the University of California

UC San Diego

UC San Diego Previously Published Works bannerUC San Diego

Ca(2+)-independent cytosolic phospholipase A2 from macrophage-like P388D1 cells. Isolation and characterization.

  • Author(s): Ackermann, EJ;
  • Kempner, ES;
  • Dennis, EA
  • et al.
Abstract

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.

Many UC-authored scholarly publications are freely available on this site because of the UC's open access policies. Let us know how this access is important for you.

Main Content
For improved accessibility of PDF content, download the file to your device.
Current View