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Journal of Lipid Research, Vol. 41, 1222-1230, August 2000
Copyright © 2000 by Lipid Research, Inc.

Original Article

Purification, characterization, and cDNA sequencing of cytosolic phospholipase A₂ from equine neutrophils

Correspondence to: Pontus K. A. Larsson Forsell

It has been demonstrated that equine neutrophils, but not eosinophils, require exogenous arachidonic acid for calcium ionophore A23187-induced leukotriene synthesis. Because cytosolic phospholipase A₂ (cPLA₂) plays an essential role in leukotriene formation in leukocytes, we investigated the presence of a functional cPLA₂ in equine neutrophils. To determine whether cPLA₂ from neutrophils was catalytically active, we purified the enzyme >6,500 fold with 3% recovery from equine neutrophils. The full-length cDNA sequence encoded a 749-amino acid protein. The deduced amino acid sequence demonstrated 95% identity with human and mouse cPLA₂, as well as 83 and 73% identity with chicken and zebra fish cPLA₂ protein, respectively. The equine cPLA₂ possessed some properties that distinguished the equine enzyme from the human enzyme. First, the enzyme activity of the equine cPLA₂ was differently influenced by cations as compared with the human cPLA₂. Second, the equine neutrophil cPLA₂ migrated as an approximately 105-kDa protein, in comparison with human cPLA₂ which migrated as a 110-kDa protein. A difference between equine neutrophils and eosinophils in the degree of phosphorylation of the cPLA₂ protein was observed. Thus, the cPLA₂ protein from eosinophils was constitutively phosphorylated, while the cPLA₂ protein from neutrophils was unphosphorylated.

In summary, these results demonstrate that equine neutrophils indeed express an active cPLA₂ protein but that there is a difference in the degree of phosphorylation of the cPLA₂ protein between equine neutrophils and eosinophils. This difference might explain the difference between the two cell types in the capacity to produce leukotrienes from endogenous substrate.—Larsson Forsell, P. K. A., Å. Lindberg, S. Karlsson, J. Å. Lindgren, and H-E. Claesson. Purification, characterization, and cDNA sequencing of cytosolic phospholipase A₂ from equine neutrophils. J. Lipid Res. 2000. 41: 1222;–1230.

Supplementary key words: phospholipase A₂, arachidonic acid, leukotriene, equine, neutrophils, eosinophils, phosphorylation

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