Solubilization and characterization of a neutral-active, calcium-dependent, phospholipase A2 from rabbit heart and isolated chick embryo myocytes

Abstract

Homogenates of rabbit heart hydrolyzed the phospholipids of autoclaved E. coli maximally at pH 7.0 with 5.0 mM added CaCl2; EDTA was a potent inhibitor. More than 70% of the homogenate phospholipase A activity was sedimented at 100 000 x g. Homogenates dialyzed to pH 3.0 had a similar pH optima, but required less than 1.0 mM added CaCl2 for optimal activity. Sulfuric acid extraction of whole heart, and other rabbit organs, solubilized a calcium-dependent phospholipase A that was maximally active at pHs 7.0 to 7.5. Myocardial extracts were as active (approx. 60 nmol/h/mg) as acid extracts from rabbit lung, liver and kidney. Phospholipase(s) A with similar properties were solubilized by acid extraction of 12-day-old chick embryo myocytes (10 nmol/h/mg). Regardless of origin, the phospholipases A were absolutely specific for release of fatty acid from the 2-acyl position of phospholipids. Activity was inhibited by (i) pretreatment with p-bromo-phenacylbromide; (ii) the anesthetics, dibucaine and chlorpromazine; and (iii) the nonsteroidal antiinflammatory agents, indomethacin, ibuprofen and meclofenamate. Aspirin and dexamethasone had little or no effect on enzymic activity.