Purification, characterization, and cDNA sequencing of cytosolic phospholipase A(2) from equine neutrophils.
It has been demonstrated that equine neutrophils, but not eosinophils, require exogenous arachidonic acid for calcium ionophore A23187-induced leukotriene synthesis. Because cytosolic phospholipase A(2) (cPLA(2)) plays an essential role in leukotriene formation in leukocytes, we investigated the presence of a functional cPLA(2) in equine neutrophils. To determine whether cPLA(2) 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 cPLA(2), as well as 83 and 73% identity with chicken and zebra fish cPLA(2) protein, respectively. The equine cPLA(2) possessed some properties that distinguished the equine enzyme from the human enzyme. First, the enzyme activity of the equine cPLA(2) was differently influenced by cations as compared with the human cPLA(2). Second, the equine neutrophil cPLA(2) migrated as an approximately 105-kDa protein, in comparison with human cPLA(2) which migrated as a 110-kDa protein. A difference between equine neutrophils and eosinophils in the degree of phosphorylation of the cPLA(2) protein was observed. Thus, the cPLA(2) protein from eosinophils was constitutively phosphorylated, while the cPLA(2) protein from neutrophils was unphosphorylated. In summary, these results demonstrate that equine neutrophils indeed express an active cPLA(2) protein but that there is a difference in the degree of phosphorylation of the cPLA(2) 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.
Forsell
P K
PK
Department of Medical Biochemistry and Biophysics, Division of Physiological Chemistry II, Karolinska Institutet, S-171 77 Stockholm, Sweden.
Lindberg
A
A
Karlsson
S
S
Lindgren
J A
JA
Claesson
H E
HE
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D000001:Calcimycin / Q000494:pharmacology; D002850:Chromatography, Gel; D003600:Cytosol / Q000201:enzymology; D018076:DNA, Complementary / Q000737:chemistry*; D004804:Eosinophils / Q000201:enzymology; D015870:Gene Expression; D006736:Horses / Q000097:blood* / Q000235:genetics; D006801:Humans; D007476:Ionophores / Q000494:pharmacology; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000201:enzymology*; D010741:Phospholipases A / Q000097:blood / Q000737:chemistry / Q000235:genetics*; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
D018076:DNA, Complementary; D007476:Ionophores; D000001:Calcimycin; D010741:Phospholipases A