Characterisation of soluble and cell associated phospholipase A2 from rheumatoid synovial fluid.
The hydrolysis of radiolabelled Escherichia coli phospholipids, and micellar dispersions of phosphatidylethanolamine and phosphatidylcholine, were used to characterise the phospholipase A2 activity in synovial fluid from patients with rheumatoid arthritis. Cell free fractions of synovial fluid contain a phospholipase A2 enzyme that preferentially releases [14C]oleic acid from E coli biomembranes (specific activity 291.3 (SEM 27.6) pmol/min/mg). This enzyme requires calcium and is optimally active at neutral pH. Purified dispersions of phosphatidylethanolamine are also readily degraded by the soluble enzyme, but it is not active against phosphatidylcholine. The substitution of [14C]oleic acid by [3H]arachidonic acid for the labelling of E coli allowed differentiation between the soluble phospholipase A2 and the cell associated phospholipase A2 present in sonicates of mononuclear cells and neutrophils from peripheral blood and synovial fluid. The cell associated phospholipase A2 preferentially releases [3H]arachidonic acid from E coli cardiolipin. In this paper the phospholipid substrate specificity of phospholipase A2 from rheumatoid synovial fluid, the optimal assay conditions for its detection, and a standardised expression of activity in terms of pmol per minute per mg of protein are established.
Department of Medicine, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, North Carolina.
H
H
Gonzalez-Buritica
D M
DM
Smith
R A
RA
Turner
D001172:Arthritis, Rheumatoid / Q000201:enzymology*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000201:enzymology; D009504:Neutrophils / Q000201:enzymology; D010740:Phospholipases / Q000378:metabolism*; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D012995:Solubility; D013379:Substrate Specificity; D013582:Synovial Fluid / Q000166:cytology / Q000201:enzymology*
D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2