The 85-kDa, arachidonic acid-specific phospholipase A2 is expressed as an activated phosphoprotein in Sf9 cells.
A human, 85-kDa arachidonoyl-specific, cytosolic phospholipase A2 was expressed using the baculovirus-insect cell expression system. Expression resulted in the production of an active protein which consisted of approximately 3% of the total protein in the host Spodoptera frugiperda (Sf9) cells at 67 h after infection. The phospholipase A2 was purified to apparent homogeneity and exhibited calcium-dependent phospholipase A2 activity with a specific activity of 8 mumol/min/mg protein, as well as calcium-independent lysophospholipase activity with a specific activity of 17 mumol/min/mg protein. The phospholipase A2 was expressed as a phosphoprotein and was primarily phosphorylated on serine residues. Phosphatase treatment of the recombinant phospholipase A2 resulted in dephosphorylation of the enzyme and a 63% decrease in phospholipase A2 activity. This decrease in activity is similar in magnitude to the decrease in activity observed with phosphatase-treated phospholipase A2 from stimulated mammalian cells. These data demonstrate that the 85-kDa phospholipase A2 is expressed as an activated phosphoprotein in Sf9 cells.
de Carvalho
M S
MS
Department of Pediatrics, National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado 80206.
McCormack
F X
FX
Leslie
C C
CC
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology; D002460:Cell Line; D002848:Chromatography, DEAE-Cellulose; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000201:enzymology; D004789:Enzyme Activation; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D009036:Moths; D010710:Phosphates / Q000378:metabolism; D010741:Phospholipases A / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D054467:Phospholipases A2; D010750:Phosphoproteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D014162:Transfection
D010710:Phosphates; D010750:Phosphoproteins; D011994:Recombinant Proteins; D016718:Arachidonic Acid; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium