Phospholipase A2 in human placental serum.

Intervillous blood was collected from term placentae at delivery, and sera were tested for phospholipase A2 under various experimental conditions. Enzyme activity was found to develop upon extended storage in the cold or at 37 degrees C. The enzyme is reversibly inhibited by dithiothreitol, requires Ca++ ions for activity, and tolerates various detergents. The apparent molecular weight is 42 kDa. In all these parameters the serum enzyme behaves similar to the 42 kDa phospholipase A2 which we recently purified to homogeneity from thoroughly washed placental tissue. Serum phospholipase A2 appears to be generated by proteolytic processing from a slightly larger inactive precursor which was detected immunochemically. Most likely this protein originates from fetal cells and may be released by membrane damage. We conclude that both placental serum and tissue harbour a novel type of phospholipase A2 which is distinct from cytosolic and secretory phospholipases A2. Preference for arachidonate containing substrate suggests a role in eicosanoid production within gestational tissues.

A novel phospholipase A2 from human placenta.

A major soluble phospholipase A2 of human term placenta was characterized and purified about 15,000-fold to homogeneity. The apparent molecular mass as determined in SDS/polyacrylamide gels is 42 kDa. The enzyme is inhibited by dithiothreitol indicating the presence of disulphide bridges which are essential for activity. Studies with known phospholipase A2 inhibitors revealed no immediate relationship to either secretory or cytosolic phospholipases A2. The placental enzyme prefers liposomes of phosphatidylcholine and has a distinct preference for arachidonic acid in the sn-2 position. It tolerates various detergents. Roughly 10 microM Ca2+ is required for activity, but it cannot be replaced by Mg2+ or Mn2+; Zn2+, Cu2+ and Fe3+ are inhibitory. In immunoblots, the placental enzyme was not detected by two separate antisera specific for type-II phospholipases A2 but reacted very weakly with antisera directed against cytosolic phospholipase A2. From these data we suggest that this enzyme is a novel form of phospholipase A2 which may be involved in arachidonic acid mobilization both during the course of pregnancy and at parturition.

Membranes exert indirect negative control on phospholipase A2 in human placenta.

Phospholipase A2 (PLA2) activity of human term placenta is distributed about equally between cytosol and membranes. The latter activity was detached by treating membranes with EGTA, but this extraction also released inhibitory protein, which complicated the assay and has probably often led to underestimation of such PLA2. Varying the substrate concentration, we found that large amounts of liposome substrate relieve PLA2 suppression in the extract. This suggests substrate depletion by the inhibitory protein as the mechanism by which PLA2 enzymes are negatively controlled in placenta. Membrane-bound PLA2 was purified about 700-fold and appeared to be one enzyme species (PLA2-M). By contrast, cytosolic PLA2 activity could be fractionated into four separate fractions, one of which was further purified (PLA2-S1). As judged on the basis of a variety of biochemical properties, PLA2-M and PLA2-S1 seem to be identical enzyme forms. They are distinct from the class of pancreas/venom-type phospholipases A2.