The action of phospholipase A 2 from Vipera berus on mixed monolayers of L-α-didecanoylphosphatidylcholine and cholesterol

Abstract

A study is made of the enzymatic lipolysis by phospholipase A 2 of Vipera berus in monomolecular films of mixtures of lipids (L-α-didecanoylphosphatidylcholine and cholesterol) on an aqueous subphase of pH 8, in order to imitate physiological conditions. The influence on the rate of lipolysis of the composition of the mixed film, the surface pressure of the film and the amount of injected enzyme are investigated. Compression isotherms of L-α-didecanoylphosphatidylcholine and cholesterol were made; similar results to those of Joos and Delem being obtained. Non-interaction at low pressures and slight interaction at higher pressures were observed. As for any other short-chain compounds, the results present little or no deviation from the additivity rule. The rate of lipolysis was tested at different surface pressures, for a mixture of the lipids in the proportion 0.5/0.5, by injecting phospholipase of Vipera berus, at a temperature of 25°C. The optimum surface pressure was observed to be 29 dyn cm −1. Subsequently a study was made to find out how the rate of the lipolytic process varied with the amount of injected enzyme at 25°C, using the same mixture and in the same molar fraction at a pressure of 29 dyn cm −1. A linear increase in the reaction rate with the amount of enzyme injected was observed, and this had to be stabilized by using amounts of enzyme > 3.2 μg thus resulting in an optimum amount of 2.56 μg. As the optimum conditions of surface pressure and amount of enzyme were already known, the influence of the composition of the mixed film on the rate of lipolysis could be studied. However, from the results obtained, an optimum proportion for the lipolytic process could not be found. A detailed interpretation of the results obtained is given.