Phospholipase A2 action on planar lipid bilayers generates a small, transitory current that is voltage independent.
Addition of either bee venom or Trimeresurus flavoviridis phospholipase A2 (PLA2) to the solution bathing the front side of a voltage-clamped, planar lipid bilayer consistently produced a transitory current lasting approximately 100 s. This current is consistent with anions moving through the membrane to the rear side. The peak current is independent of holding potential. PLA2 activity on phospholipid membranes not only produced a current but also led to membrane rupture within 300 s. The current depends on Ca2+ and lipid type. Addition of PLA2 in the absence of Ca2+ or to membranes made of nonsubstrate lipids (e.g., glycerol monooleate or lysophosphatidylcholine) produced no current and did not break the bilayer. Peak current height, signal decay time, and time to membrane rupture all depended on PLA2 dose, whereas total charge produced was constant. This current does not flow through ion channels because there are no channels present and the current is not voltage dependent. The evidence is consistent with the hypothesis that the current is generated by the movement of ionized fatty acid produced by PLA2 action. These results demonstrate a simple method to measure enzyme activity in the presence of different substrates and varied environmental conditions.
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Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
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D000818:Animals; D001514:Bee Venoms; D001921:Brain; D017839:Crotalus; D004553:Electric Conductivity; D007473:Ion Channels; D008051:Lipid Bilayers / Q000737:chemistry* / Q000378:metabolism; D008564:Membrane Potentials; D008954:Models, Biological; D010713:Phosphatidylcholines / Q000737:chemistry / Q000378:metabolism; D010714:Phosphatidylethanolamines / Q000737:chemistry / Q000378:metabolism; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D013379:Substrate Specificity
D001514:Bee Venoms; D007473:Ion Channels; D008051:Lipid Bilayers; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D010741:Phospholipases A; D054467:Phospholipases A2