title: Metal ion and salt effects on the phospholipase A2, lysophospholipase, and transacylase activities of human cytosolic phospholipase A2.
abstract: 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.
authors: Department of Chemistry, University of California, San Diego, La Jolla 92093-0601.
authors: L J
authors: LJ
authors: Reynolds
authors: L L
authors: LL
authors: Hughes
authors: A I
authors: AI
authors: Louis
authors: R M
authors: RM
authors: Kramer
authors: E A
authors: EA
authors: Dennis
mesh_terms: 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*
chemical_list: D002413:Cations, Divalent; D008244:Lysophosphatidylcholines; D008670:Metals; D011994:Recombinant Proteins; D012492:Salts; D000217:Acyltransferases; D010741:Phospholipases A; D054467:Phospholipases A2; D008245:Lysophospholipase; D002118:Calcium