Activation of phospholipase A2 on lipid bilayers.

So far, three phospholipases A2 that display activation kinetics during the time course of hydrolysis of DPPC LUV have been found to undergo a fluorescence change coincident with the activation: the monomer (AppD49) and the dimer enzymes from A. piscivorus piscivorus and the dimer enzyme from C. atrox. The porcine pancreatic enzyme produces similar time courses of hydrolysis but does not display a concurrent fluorescence change. It is assumed that other phospholipases A2 will behave similarly in terms of the hydrolysis reaction. Which enzymes respond with a similar change in intrinsic fluorescence during the time course may well depend on the position of tryptophan residues and the amino acid sequence. Even though a given phospholipase A2 may not change its fluorescent properties on activation, the simultaneous monitoring of the hydrolysis reaction and the fluorescence of probes of the bilayer structure can be done with any phospholipase A2. A variety of probes exist which are sensitive to slightly different membrane properties and could be used as described here for TMA-DPH. For example, 1,3-dipyrenylpropane is sensitive to the apparent microviscosity of the bilayer is terms of the ability of molecules to translationally diffuse in the membrane. 6-Palmitoyl-2-[[2-(trimethylammonio)ethyl]methylamino]naphthalene chloride is sensitive to the ability of a molecule to rotate in the bilayer and displays large changes in its steady-state fluorescence as the anisotropy of the bilayer changes. 6-Propionyl-2-(dimethylamino)naphthalene is sensitive to the polarity and degree of hydration of its environment. Finally, a compound titled NK-529 has recently been introduced that apparently monitors the lateral phase separation of fatty acids in the bilayer. The fact that activation of phospholipase A2 can be monitored during the time course of hydrolysis of DPPCLUV makes this system an excellent choice for studying the mechanisms of activation and possible effects of various activators and inhibitors. The experimental system described here provides a way to determine whether such regulators exert their effects through alterations of the properties of the membrane and/or the enzyme. Importantly, this system allows one to seek temporal correlations of the various events in the process.