Purification and characterization of cytosolic phospholipase A₂ activities from canine myocardium and sheep platelets

This chapter describes the assay method, purification procedures, and properties of the spleen phospholipases A₂ enzymes. As an optimal molar ratio depends on the detergent-phospholipid combination, the ratio is determined for each set of detergent and phospholipid. The solvents are evaporated under nitrogen, and then the samples are further dried in vacuo for about 30 min. An appropriate amount of the buffer is added, and the tubes are vortexed for 2 min at about 37°. The key step of the purification procedures is reversed-phase high-performance liquid chromatography (HPLC). The PLA₂ preparations (PLA₂ S-1 and PLA₂ M) purified from the two sources are homogeneous as judged by sodium dodecyl sulfate (SDS)–gel electrophoresis and analytical HPLC. Both enzymes migrates on SDS gels as a single protein band to the same position as rat pancreatic PLA₂. The apparent molecular weight of both enzymes is estimated to be 13,600, in agreement with the weights calculated from the amino acid sequences. The amino acid compositions of PLA₂ S-1 and pancreatic PLA₂ are similar and the peptide maps and sequences of the amino-terminal 32 residues of the two enzymes are identical. PLA₂ S-1 is immunochemically identical to rat pancreatic PLA₂, whereas there is no immunochemical similarity between PLA₂ M and pancreatic PLA₂. An antipancreatic PLA₂ antibody do not recognize PLA₂ M, and conversely an anti-PLA₂ M antibody do not recognize the pancreatic PLA₂.