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.

Signal transduction pathways leading to arachidonic acid release from neutrophilic HL-60 cells. The involvement of G protein, protein kinase C and phospholipase A2.

Arachidonic acid release from undifferentiated and neutrophilic HL-60 cells was studied. In neutrophilic cells it was stimulated by N-formyl-Met-Leu-Phe and mastoparan by a mechanism involving Gi protein and phospholipase C and was largely dependent on diacyglycerol lipase. Maximum release from both cell types was achieved with fluoride and required cellular energy. Inhibitor studies suggest that arachidonic acid release by fluoride stimulation leads to phospholipase A2 activation with signal transduction involving phospholipase C and protein kinase C. Only neutrophilic cells responded to phorbol ester if Ca(2+)-ionophore was simultaneously present but this effect was abolished by extended treatment with phorbol ester. Thus, protein kinase C plays a major role in highly stimulated neutrophilic cells. These cells are differently equipped with protein kinase C isoenzymes compared with undifferentiated cells. In contrast, both cell types contain similar levels of type II and cytosolic phospholipases A2, the former being by far the more prevalent.

Annexins and phospholipase A2 inhibition.

Annexins of human placenta have been purified and characterized. In addition to annexins I to VI and II complex, two novel species of 45 and 68 kDa were obtained. Annexins V and II are most abundant. Phospholipase A2 inhibitory activity of annexin V is low in contrast to that of annexins I to VI, and it is best for annexin II complex. In vitro, annexins bind to liposomes to extents which depend on the type of phospholipid used. This induces liposome aggregation whereby Mix, PI, and PC liposomes preferably aggregate. This hinders PLA2 from its access to the substate. Our data suggest that substrate-depletion by annexins is rather the result of liposome cross-bridging than pure liposome surface coating.

A series of annexins from human placenta and their characterization by use of an endogenous phospholipase A2.

Membranes from human placenta contain proteins which inhibit the activity of phospholipases A2 by binding to phospholipid thus impeding substrate availability. We used unilamellar mixed liposomes and a partially purified cytosolic phospholipase A2 from placenta for characterizing this substrate-depleting activity. A major portion of these inhibitory proteins was released by extracting washed membranes with a Ca+(+)-chelator. Biochemical fractionation and systematic analysis resulted in the unequivocal identification of a series of annexin proteins. We describe a straightforward procedure which allows to obtain 8 annexins from placenta either in pure form or as a mixture of two annexins. One of them was obtained in two forms which had the same molecular mass of 68 kDa but differed in charge. We also present suggestive evidence for a novel annexin I-related polypeptide of Mr 45,000 which is an excellent in vitro substrate for protein kinase C. We estimate that about 2% of the total placental membrane proteins are annexins. For achieving half inhibition of phospholipase A2 activity with pure annexins, up to a 6.5-fold difference in the amounts of protein was observed when calculated on a molar basis. This suggests specificity of individual annexin species.

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.

A rabbit reticulocyte factor which stimulates protein synthesis in several mammalian cell-free systems.

Rabbit reticulocyte lysate post-ribosomal supernatant is shown to stimulate protein synthesis in a variety of mammalian cell-free systems, particularly the less efficient systems, such as those from mouse liver, HeLa cells and heat-shocked L cells. This stimulation reflects an increase in the rate of initiation, and is not due to the presence of globin mRNA. The stimulatory activity is unstable to purification, but some conditions favouring stability have been identified and partial purification has been achieved. It is free of eIF-2, but possesses eIF-2B activity. Its purification properties suggest that it is distinct from previously characterized initiation factors, including eIF-2/eIF-2B complex, and its possible relationship to known initiation factors is discussed.