CHARACTERIZATION OF PHOSPHOLIPASE A(2) ACTIVITY ENRICHED IN THE NERVEGROWTH CONE

Nerve growth cones isolated from fetal rat brain exhibit in their cyto sol a robust level of phospholipase A(2) activity hydrolyzing phosphat idylinositol (PI) and phosphatidylethanolamine (PE) but not phosphatid ylcholine (PC). Western blot analysis with an antibody to the well-cha racterized cytosolic phospholipase A(2) (mel wt 85,000) reveals only t race amounts of this PC- and PE-selective enzyme in growth cones. By g el filtration on Superose 12, growth cone phospholipase A(2) activity elutes essentially as two peaks of high molecular mass, at similar to 65 kDa and at well over 100 kDa. Anion exchange chromatography complet ely separates a PI-selective from a PE-selective activity, indicating the presence of two different, apparently monoselective phospholipase A(2) species. The PI-selective enzyme, the predominant phospholipase A (2) activity in whole growth cones, is enriched greatly in these struc tures relative to their parent fractions from fetal brain. This phosph olipase A(2) is resistant to reducing agents and is found in the cytos ol as well as membrane-associated in the presence of Ca2+. However, it s catalytic activity is Ca2+-independent regardless of whether the enz yme is associated with pure substrate or mixed-lipid growth cone vesic les. The PE-selective phospholipase A(2) in growth cones was studied i n less detail but shares with the PI-selective enzyme several properti es, including intracellular localization, the existence of cytosolic a nd membrane-associated forms, and Ca2+ independence. Our data indicate growth cones contain two high-molecular-weight forms of phospholipase A(2) that share many properties with known, Ca2+-independent cytosoli c phospholipase A(2) species but that appear to be monoselective for P I and PE, respectively. In particular, the PI-selective enzyme may rep resent a new member of the growing family of cytoplasmic phospholipase s A(2). The enrichment of the PI-selective phospholipase A(2) in growt h cones suggests it plays a major role in the regulation of growth con e function.