Cytosolic phospholipase A2 from human monocytic cells: characterization of substrate specificity and Ca(2+)-dependent membrane association.
A cytosolic phospholipase A2 (PLA2) has been purified to homogeneity from the human monocytic tumour cell line THP-1 by a combination of ion-exchange, heparin-agarose and hydrophobic-interaction chromatography and non-denaturing PAGE. The purified PLA2 exhibited a molecular mass of 100 kDa on SDS/PAGE, whereas non-denaturing PAGE showed a molecular mass of about 80 kDa. Investigation of the substrate specificity of PLA2 revealed no preference for arachidonic acid- over linoleic acid-containing phospholipids when labelled THP-1 membranes were used as substrate. Treatment of THP-1 cells for 1-72 h with the phorbol ester phorbol 12-myristate 13-acetate (PMA) led to a stable enzyme activation, which was also found after partial purification of PLA2 from PMA-stimulated THP-1 cells. The properties of the partially purified PLA2 from PMA-stimulated cells were compared with those of the PLA2 from unstimulated cells. Both enzymes showed an identical Ca(2+)-dependent enzyme activation (EC50 = 300 nM Ca2+). A significant difference was found in the Ca(2+)-dependent membrane association of the enzymes obtained from the two sources. PLA2 from PMA-stimulated cells showed membrane association of PLA2 activity at significantly lower Ca2+ concentrations (100-300 nM) compared with the PLA2 from unstimulated THP-1 cells (300-500 nM). The altered Ca(2+)-dependency was due to a stable modification of the enzyme. Changes in the ability of PLA2 to associate with cellular membranes may thus be regarded as an important molecular mechanism underlying the increase in fatty acid metabolism observed in activated cells.
Rehfeldt
W
W
Institute of Molecular Pharmacology, Medical School Hannover, Federal Republic of Germany.
Resch
K
K
Goppelt-Struebe
M
M
D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D003600:Cytosol / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D008970:Molecular Weight; D009000:Monocytes / Q000201:enzymology*; D010741:Phospholipases A / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D054467:Phospholipases A2; D013379:Substrate Specificity; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D010741:Phospholipases A; D054467:Phospholipases A2; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium