A NOVEL CYTOSOLIC CALCIUM-INDEPENDENT PHOSPHOLIPASE-A(2) CONTAINS 8 ANKYRIN MOTIFS

We report the purification, molecular cloning, and expression of a nov el cytosolic calcium-independent phospholipase A(2) (iPLA(2)) from Chi nese hamster ovary cells, which lacks extended homology to other phosp holipases. iPLA(2) is an 85-kDa protein that exists as a multimeric co mplex of 270-350 kDa with a specific activity of 1 mu mol/min/mg. The full-length cDNA clone encodes a 752-amino acid cytoplasmic protein wi th one lipase motif (GXS(465)XG) and eight ankyrin repeats. Expression of the cDNA in mammalian cells generates an active 85-kDa protein. Mu tagenesis studies show that Ser(465) and the ankyrin repeats are requi red for activity. We demonstrate that iPLA, selectively hydrolyzes the sn-2 over sn-l fatty acid by 5-fold for 1,2-dipalnitoyl phosphatidylc holine in a mixed micelle. Moreover, we found the fatty acid preferenc e at the sn-2 position to be highly dependent upon substrate presentat ion. However, iPLA, does have a marked preference for 1,2-dipalnitoyl phosphatidic acid presented in a vesicle, generating the lipid second messenger lysophosphatidic acid. Finally the enzyme is able to hydroly ze the acetyl moiety at the sn-2 position of platelet-activating facto r.