Regulation of phospholipase A2 activity by different lipid water interfaces

Abstract

The activity of pancreatic phospholipase A2 (EC 3.1.1.4) is controlled not only by the architecture of the catalytic site, but is also strongly dependent on the penetrating power of the interface recognition site and the packing density of the lipid water interface. The influence of the latter two factors on the interface activity has been investigated using chemically modified phospholipases A2 in which the NH2 terminal L-Ala8 has been replaced by DL-[3-13C]Ala, or in which the polypeptide chain has been elongated with DL-[3-13C]Ala. The [DL-(3-13C)Ala8]phospholipase A2 could be resolved into the pure diastereoisomers, [D-(3-13C)Ala8]phospholipase A2 and [L-(3-13C)Ala8]phospholipase A2 by elution on Sephadex G-100 in the presence of a micellar lipid water interface, as well as by conventional ion exchange chromatography on carboxymethylcellulose. Similar procedures did not effect, however, a separation of DL-[3-13C]Ala7-phospholipase A2 into their respective diastereoisomers, indicating the strategic role of the NH2-terminal L-Ala8 residue in the interaction process between the enzyme and lipid water interfaces. Kinetic experiments using various micellar short chain lecithins revealed the apparent absence of an interface recognition site in [D-(3-13C)Ala8]- and DL-[3-13C]Ala7- phospholipase A2 while these proteins still possess considerable enzymatic activity toward monomeric substrates. In contrast, however, kinetic experiments using monomolecular surface films, allowing a continuous change in surface density of the substrate molecules revealed that [D-(3-13C)Ala8]- and DL-[3-13C]Ala7- phospolipase A2 at low surface pressures possess about 60 and 30% of the interface activity of native phospholipase A2 respectively. These results therefore suggest that the modified phospholipases A2 do possess an interface recognition site although less powerful as compared to that of the native enzyme, enabling the estimation of the surface density of micellar short chain lecithins.