[36] Purification of mammalian nonpancreatic extracellular phospholipases A2

Publisher Summary This chapter describes the methods of purifying phospholipases A 2 from supernatants of rat activated platelets, human synovial fluid, and rat peritoneal fluid. The most distinct property commonly observed in mammalian nonpancreatic extracellular phospholipases A 2 is a high affinity for heparin. Heparin-Sepharose affinity chromatography is therefore a powerful tool for the purification of these enzymes. Extracellular phospholipase A 2 released from rat platelets is purified about 770-fold to near homogeneity by the sequential use of column chromatography on heparin-Sepharose and TSK gel G2000SW. Lysophosphatidylserine-specific lysophospholipase, which is also released from rat platelets, is separated from phospholipase A 2 by chromatography on heparin-Sepharose. Human rheumatoid synovial fluid phospholipase A 2 also shows high affinity for heparin. The enzyme is purified to near homogeneity by sequential use of heparin-Sepharose, butyl-Toyopearl, and reversed-phase high-performance liquid chromatography (HPLC). Pooled human synovial fluid from patients with rheumatoid arthritis is loaded onto a Sepharose CL-4B precolumn and eluted with 50 mM Tris-HCl (pH 7.4) containing 0.15 M NaCl. Extracellular phospholipase A 2 , which shares a common structure with the enzyme released from activated rat platelets, is detected in the peritoneal cavity of rats injected intraperitoneally with casein. The enzyme can be purified about 14,000-fold to near homogeneity from rat peritoneal fluid without using heparin-Sepharose chromatography.