Metal ion and salt effects on the phospholipase A2, lysophospholipase, and transacylase activities of human cytosolic phospholipase A2.
Human cytosolic phospholipase A2 (cPLA2) is an arachidonic acid specific enzyme which may play a role in arachidonic acid release, eicosanoid production, and signal transduction. The PLA2 activity of this enzyme is stimulated by microM levels of Ca2+. Using a pure recombinant enzyme, we have confirmed that cPLA2 is not absolutely dependent on Ca2+, since Sr2+, Ba2+ and Mn2+ also gave full enzyme activity. Heavy metals, in contrast, inhibited enzyme catalysis suggesting the involvement of an essential cysteine residue. In the absence of Ca2+, high salt concentrations overcame the requirement for divalent metals, indicating that Ca2+ is not required for PLA2 catalytic activity. cPLA2 also displays a lysophospholipase (lyso PLA) activity with lysophosphatidylcholine micelles as a substrate. Unlike the PLA2 activity, the lyso PLA activity toward these micelles is not stimulated by Ca2+. However, upon the addition of glycerol or Triton X-100 to the assay, Ca2+ activation is observed, indicating that substrate presentation can affect the apparent Ca2+ dependence. Glycerol was found to be a potent stimulator of lyso PLA activity and specific activities up to 50 mumol min-1 mg-1 were observed. In addition to the PLA2 and lyso PLA activities, we report that cPLA2 displays a relatively low, CoA-independent transacylase activity which produces phosphatidylcholine from lysophosphatidylcholine substrate. The observation of this novel transacylase activity is consistent with the formation of an acyl-enzyme intermediate.
Reynolds
L J
LJ
Department of Chemistry, University of California, San Diego, La Jolla 92093-0601.
Hughes
L L
LL
Louis
A I
AI
Kramer
R M
RM
Dennis
E A
EA
D000217:Acyltransferases / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology; D002413:Cations, Divalent; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D008244:Lysophosphatidylcholines / Q000378:metabolism; D008245:Lysophospholipase / Q000378:metabolism*; D008670:Metals / Q000494:pharmacology*; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D011994:Recombinant Proteins / Q000378:metabolism; D012492:Salts / Q000494:pharmacology*
D002413:Cations, Divalent; D008244:Lysophosphatidylcholines; D008670:Metals; D011994:Recombinant Proteins; D012492:Salts; D000217:Acyltransferases; D010741:Phospholipases A; D054467:Phospholipases A2; D008245:Lysophospholipase; D002118:Calcium