Spontaneous domain formation of phospholipase A 2 at interfaces: Fluorescence microscopy of the interaction of phospholipase A 2 with mixed monolayers of lecithin, lysolecithin and fatty acid

Abstract

Fluorescence microscopy has recently been proven to be an ideal tool to investigated the specific interaction of phospholípase A 2 with oriented substrate monolayers. Using a dual labeling technique, it could be shown that phospholipase A 2 can specifically attack and hydrolyze solid analogous l-α-DPPC domains. After a critical extent of monolayer hydrolysis the enzyme itself starts to aggregate forming regular shaped protein domains (Grainger et al. (1990) Biochim. Biophys. Acta 1023. 365–379). In order to confirm that the existence of hydrolysis products in the mononlayer is necessary for the observed aggregation of phospholipase A 2, mixed monolayers of d- and l-α-DPPC, l-α-lysoPPC an palmitic acid in different ratios were examined. The phase behavior and the interaction of these films with phospholipase A 2 were directly visualized with an epifluorescence microscope. Above a certain critical concentration of lysolecithin and palmitic acid in the monolayer, compression of these mixed films leads to phase separation and formation of mixed domains of unknown composition. Their high negative charge density is evidenced by preferential binding of a cationic dye to these phase-separated areas. Introduction of fluorescence-labeled phospholipase A 2 underneath these mixed domains results in rapid binding of the protein to the domains without visible hydrolytic activity, regardless of whether the l-form of the d-form of the DPPC were used In binary mixtures, only those with DPPC/palmitic acid show formation of phase-separated areas which can be specifically targeted by phospholipase A 2 leading to a rapid formation (within 2 min) of protein domains. Experiments with pyrenedecanoic acid containing monolayers give the first direct evidence that acid is located above the enzyme domains. These results show that a locally high negative charge density of the phase-separated domains is one of the prerequisites for the binding of phospholipase A 2. In addition, however, small amounts of d- or l-α-DPPC headgroups within the domains of the monolayer seen to be necessary for recognition followed by fast binding of the protein to the domains. This is confirmed by experiments with mixed monolayers of diacetylene carboxylic acid and d-ga-DPPC. The acid - immiscible with lecithn - forms well defined pure acid domains in the monolayer. While the cationic dye can be docked rapidly to these phase separated areas, no preferential enzyme binding and thus no protein domain formation below these acid domains can be induced.