Thermophilic phospholipase A(2) in the cytosolic fraction from the archaeon Pyrococcus horikoshii

Hyperthermophilic archaeon Pyrococcus horikoshii produced phospholipase A(2 ) in a cytosolic fraction. The enzyme displayed optimal activity at 90 degr eesC and pH 7 and preferentially hydrolyzed sn-2 fatty acids in the followi ng order: linoleoyl > oleoyl > arachidoyl phosphatidylcholine. Phospholipas e A(2) had similar activities toward L-alpha -1-palmitoyl-2-arachidoyl deri vatives of phosphatidylcholine and phosphatidylethanolamine. Phospholipase A(2) activity was unaffected by the addition of 0.0001-1 mM calcium, but wa s inhibited slightly by the addition of 2-10 mM calcium. The activity was e nhanced more than 5-18-fold in the presence of 3-20% (vol/vol) glycerol. Th e activity was unaffected by the addition of 1-5 mM EDTA or 0.01-20 mM dith iothreitol. A caldarchaetidic acid derivative having a molecular weight of 1544 disappeared upon incubation of the cytosolic fraction with total lipid . The phospholipase A(2) was difficult to purify by general chromatography because it existed as an aggregate. Electrophoresis was carried out using 1 0-15% polyacrylamide gels containing sodium dodecyl sulfate (SDS-PAGE). No activity of phospholipase A(2) activity was observed in the absence of prot eins less than 19 kD in size, as fractionated by SDS-PAGE.