NDLI: Purification, characterization, and cDNA sequencing of cytosolic phospholipase A2 from equine neutrophils1

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Author	Pontus K. A. Larsson Forsell Åsa Lindberg Sture Karlsson Jan Åke Lindgren Hans-Erik Claesson
Abstract	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.
ISSN	00222275
e-ISSN	15397262
DOI	10.1016/S0022-2275(20)33429-5
Journal	Journal of Lipid Research
Issue Number	8
Volume Number	41
Language	English
Publisher	Elsevier
Publisher Date	2000-01-01
Publisher Place	United States
Access Restriction	Open
Subject Keyword	Biochemistry Phospholipase A2 Arachidonic Acid Leukotriene Equine Neutrophils Eosinophils
Content Type	Text
Resource Type	Article
Subject	Cell Biology Biochemistry Endocrinology

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