Purification of a 110-kilodalton cytosolic phospholipase A2 from the human monocytic cell line U937.
The major dithiothreitol-resistant phospholipase A2 activity present in the cytosol of U937 cells has been purified greater than 200,000-fold by sequential chromatography on phenyl-5PW, heparin-Sepharose CL-6B, high-performance hydroxylapatite, TSK-gel G3000-SW, and Mono Q columns. This 110-kDa cytosolic phospholipase A2 is distinct from the relatively small (14-kDa) dithiothreitol-sensitive phospholipases A2 that are secreted from many cell types. This additional phospholipase A2 selectively hydrolyzes fatty acid at the sn-2 position of the glycerol and favors phospholipids containing arachidonic acid, which is the rate-limiting precursor for prostaglandin and leukotriene production. Interestingly, a greater than 5-fold increase in phospholipase A2 activity is noted as the calcium concentration increases from the levels found in resting cells to those observed in activated macrophages. We suggest that this enzyme and not the previously described secretory phospholipase A2 is activated by cytosolic effectors such as GTP-binding regulatory proteins and protein kinases to initiate the production of prostaglandins, leukotrienes, and platelet-activating factor. To distinguish this cytosolic enzyme from the previously described secretory ones, we suggest referring to it as cPLA2 for cytosolic phospholipase A2 and collectively referring to the secretory phospholipases A2 as sPLA2s.
Clark
J D
JD
Drug Discovery, Genetics Institute, Inc. Cambridge, MA 02140.
Milona
N
N
Knopf
J L
JL
D002118:Calcium / Q000494:pharmacology; D002460:Cell Line; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D009000:Monocytes; D010741:Phospholipases A / Q000302:isolation & purification* / Q000378:metabolism; D054467:Phospholipases A2; D013379:Substrate Specificity; D014462:Ultrafiltration
D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium