Purification, characterization, and cDNA sequencing of cytosolic phospholipase A(2) from equine neutrophils

It has been demonstrated that equine neutrophils, but not eosinophils, requ ire exogenous arachidonic acid for calcium ionophore A23187-induced leukotr iene synthesis. Because cytosolic phospholipase A(2) (cPLA(2)) plays an ess ential role in leukotriene formation in leukocytes, we investigated the pre sence of a functional cPLA(2) in equine neutrophils, To determine whether c PLA(2) from neutrophils was catalytically active, we purified the enzyme >6 ,500 fold with 3% recovery from equine neutrophils. The full-length cDNA se quence encoded a 749-amino acid protein, The deduced amino acid sequence de monstrated 95% identity with human and mouse cPLA(2), as well as 83 and 73% identity with chicken and zebra fish cPLA(2) protein, respectively. The eq uine cPLA(2) possessed some properties that distinguished the equine enzyme from the human enzyme, First, the enzyme activity of the equine cPLA(2) wa s differently influenced by cations as compared with the human cPLA(2), Sec ond, the equine neutrophil cPLA(2) migrated as an approximately 105-kDa pro tein, in comparison with human cPLA(2) which migrated as a 110-kDa protein, A difference between equine neutrophils and eosinophils in the degree of p hosphorylation of the cPLA(2) protein was observed, Thus, the cPLA(2) prote in from eosinophils was constitutively phosphorylated, while the cPLA(2) pr otein from neutrophils was unphosphorylated.jlr In summary, these results d emonstrate that equine neutrophils indeed express an active cPLA(2) protein but that there is a difference in the degree of phosphorylation of the cPL A(2) protein between equine neutrophils and eosinophils. This difference mi ght explain the difference between the two cell types in the capacity to pr oduce leukotrienes from endogenous substrate.