Inhibition of phospholipase A2 by Virasole derivation

Kinetics of phosphatidylcholine (PC) hydrolysis under the action of pancreatic phospholipase A2 IB, (EC 3.1.1.4, PLA2) in the presence of a lipophilic derivative of the antiviral drug Virazole 1-(3-((tert-butyldimethylsilyl)oxy)-4-hydroxy-5- (((4-methoxyphenyl)diphenylmethoxy)methyl)tetrahydrofuran-2-yl)-1H-1,2,4-triazole-3-carboxamide (Virazole2ЗГ) was studied. The both steps of phospholipolysis were quantitatively characterized: the binding of the enzyme to the lipid-water interface (Ks) and directly the catalytic act (Km) with the determination of the maximum reaction rate (Vmax). It was found that Virazole2ЗГ at a concentration of 0.5 μmol/ml does not affect the Ks value; on the contrary, the Michaelis constant, Km, increases by a factor of 1.8 along with the constancy of the parameter Vmax. Based on the constancy of the Ks values, it seems to be assumed that there is no inhibition of the disintegration of the enzyme-micelle complex in the presence of the effector under the studied reaction conditions. The kinetic parameters of the reaction (the increase in Km and the constancy of Vmax in the presence of Virazole2ЗГ) testify in favor of a moderate competitive inhibition of pancreatic PLA2, Ki = 65 mM, which indicates the possibility of searching for the biological activity of the anti-pancreatitis action in the series of pro-drugs of nucleoside nature.

Hydrolysis of UV-induced peroxidized phosphatidylcholine initiated by phospholipases of different substrate specificities

The activity of porcine pancreatic phospholipase A2 and the same of cobra venom toward phosphatidylcholine having different supramolecular organization and interfacial charge (micelles with sodium deoxycholate and liposomes) under UV irradiation (180–400 nm) was studied. It was shown that the UV-irradiated lipid phase is characterized by an increased index of phosphatidylcholine oxidation and the absence of a peak with a maximum of 235.5 nm, related to the presence of unsaturated bonds in the UV spectrum of docosahexaenoic acid, but retained in the presence of the antioxidant trolox. The activation of both phospholipases A2 after UV irradiation of the substrate was established, regardless of its supramolecular organization, the charge of the interfacial surface, and the substrate specificity of the enzymes. Using dynamic light scattering, 0.3 % of larger particles were found among the irradiated micelles of phosphatidylcholine. The results obtained indicate that areas of accumulation of hydroperoxidized lipids can be formed in the irradiated model membrane, which serve as a site of intensified attack for phospholipases.