Characterization of phospholipase A2 in monocytic cell lines. Functional and biochemical aspects of membrane association.
Phospholipase A2 activity was characterized in the human monocytic tumour-cell lines U937 and THP1. The enzyme showed an alkaline pH optimum and substrate specificity for arachidonoyl-phosphatidylcholine. The activation of phospholipase A2 required bivalent cations (Ca2+ greater than Mg2+ = Sr2+ greater than Ba2+). Investigation of the subcellular distribution of the enzyme revealed that the phospholipase A2 activity was shifted to the cytosol in the presence of EDTA, indicating that the association of the enzyme with the cellular membranes is Ca2+ (bivalent-cation)-dependent. Stimulation of THP1 cells for 2-4 h with the phorbol ester phorbol 12-myristate 13-acetate (PMA) activated cytosolic and membrane-bound phospholipase A2. At this time, no effect of PMA on phospholipase A2 activity was observed in the less mature U937 cells. However, when both cell lines were induced to differentiate along the monocytic pathway by a 2-3-day treatment with PMA, the cells released significant amounts of arachidonic acid and prostanoids. Compared with undifferentiated control cells, these PMA-differentiated cells showed a decrease in cytosolic phospholipase A2 activity and an increase in membrane-bound activity. Membrane-bound and cytosolic enzyme showed the same pH optimum, Ca(2+)-dependency and substrate specificity. These data indicate that membrane-bound and cytosolic phospholipase A2 activities represent one enzyme and that the membrane-bound form is the biologically active phospholipase A2.
Rehfeldt
W
W
Department of Pharmacology and Toxicology, Medical School Hannover, Federal Republic of Germany.
Hass
R
R
Goppelt-Struebe
M
M
D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology; D002471:Cell Transformation, Neoplastic; D003600:Cytosol / Q000201:enzymology; D004492:Edetic Acid / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D007951:Leukemia, Myeloid / Q000201:enzymology*; D008274:Magnesium / Q000494:pharmacology; D008565:Membrane Proteins / Q000378:metabolism / Q000502:physiology*; D010741:Phospholipases A / Q000378:metabolism / Q000502:physiology*; D054467:Phospholipases A2; D013379:Substrate Specificity / Q000187:drug effects; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D001095:Arachidonic Acids; D008565:Membrane Proteins; D016718:Arachidonic Acid; D004492:Edetic Acid; D010741:Phospholipases A; D054467:Phospholipases A2; D008274:Magnesium; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium