The Ca2(+)-sensitive cytosolic phospholipase A2 is a 100-kDa protein in human monoblast U937 cells.
Human monoblast U937 cells contain a soluble phospholipase A2 (PLA2) that is activated over the range of 150-600 nM Ca2+ and is stable only at neutral pH. We have purified this PLA2 over 34,000-fold to near homogeneity using sequential ion exchange, hydrophobic interaction, and gel filtration chromatography steps. The protein has a Mr of approximately 100,000 (by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and an isoelectric point of 5.1. Four lines of evidence indicate that this 100-kDa polypeptide represents the PLA2. (i) The intensity of staining of the 100-kDa protein was proportional to the degree of purification of PLA2 activity, (ii) the relative staining intensity of the 100-kDa protein precisely paralleled the elution profile of PLA2 activity during chromatography steps, (iii) the PLA2 activity recovered from a nondenaturing gel (greater than 60% of the total activity applied) coincided exactly with the major high molecular weight protein detected by silver staining, and (iv) monoclonal antibodies against the 100-kDa protein immunoprecipitated the PLA2. We conclude that the cytosolic PLA2 isolated from U937 cells represents a novel, high molecular weight PLA2 responding to physiological (intracellular) changes in Ca2+ concentration and therefore may play a critical role in cellular signal transduction processes and the biosynthesis of lipid mediators.
Kramer
R M
RM
Lilly Research Laboratories, Indianapolis, Indiana 46285.
Roberts
E F
EF
Manetta
J
J
Putnam
J E
JE
D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D002853:Chromatography, Liquid; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007150:Immunohistochemistry; D007700:Kinetics; D008970:Molecular Weight; D010741:Phospholipases A / Q000302:isolation & purification / Q000378:metabolism*; D054467:Phospholipases A2; D011233:Precipitin Tests
D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium