Assay of phospholipase A2 activity of synaptic membranes using a phospholipid transfer protein: stimulation by depolarization.
A phospholipid transfer protein from bovine liver was used to transfer exogenous ([14C]linoleoylphosphatidylethanolamine [14C]linoleoyl PE) into synaptic membranes and synaptosomes without modifying the lipid compositions in order to study the intrinsic activity of phospholipase A2 of the preparations. Results were compared with the conventional method in which the substrate was simply dispersed with the enzyme preparations. Liberation of [14C]linoleic acid from [14C]linoleoyl PE-preloaded synaptic membranes by the transfer protein continued almost linearly over 60 min of incubation in the presence of Ca2+. The dose-response curve of Ca2+ for the activity of phospholipase A2 obtained in the present method was slightly shifted to the left in comparison with the conventional method: Ca2+ even at the concentration of 100 microM significantly enhanced the enzyme activity. Requirement of Ca2+ for the reaction is more specific in the present method than in the conventional one. When synaptosomes were prelabeled with [14C]linoleoyl PE by the transfer protein, the liberation of [14C]linoleic acid during the incubation at 37 degrees C increased linearly over 2 min. The liberation of [14C]linoleic acid was significantly enhanced in the presence of 56 mM KCl, 50 microM veratridine and 50 microM calcium ionophore A23187. These agents did not stimulate the reaction in the absence of Ca2+.
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism*; D002413:Cations, Divalent; D002417:Cattle; D006863:Hydrogen-Ion Concentration; D008297:Male; D008563:Membrane Lipids / Q000378:metabolism*; D008564:Membrane Potentials; D008565:Membrane Proteins; D010740:Phospholipases / Q000378:metabolism*; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D049294:Phospholipid Transfer Proteins; D010743:Phospholipids / Q000378:metabolism; D051381:Rats; D013570:Synaptic Membranes / Q000201:enzymology*; D013574:Synaptosomes / Q000378:metabolism
D002352:Carrier Proteins; D002413:Cations, Divalent; D008563:Membrane Lipids; D008565:Membrane Proteins; D049294:Phospholipid Transfer Proteins; D010743:Phospholipids; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium