Purification and Characterization of Phospholipase A2 Released from Rat Platelets

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Abstract
It was found that phospholipase A2 and lysophospholipase, both of which were released from thrombin-stimulated rat platelets, had high affinity to insolubiized heparin. Phospholipase A2 released from rat platelets was purified by the sequential use of column chromatography on heparin-Sepharose and TSK gel G2000SW (high-performance liquid chromatography, HPLC). The enzyme was near homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and HPLC, and its Mr, was estimated to be 13,500. The purified enzyme was labile and lost its activity within 1 h when incubated at 37°C. Phospholipids or detergent in the solution protected the enzyme against inactivation. Phospholipase activity was inhibited by p-bromophenacylbromide, but not by diisopropylfluorophosphate or iodoacetamide. Lysophospholipase, which was also released from rat platelets, was separated from phospholipase A2 by chromatography on heparin-Sepharose.