Spontaneous domain formation of phospholipase A2 at interfaces: fluorescence microscopy of the interaction of phospholipase A2 with mixed monolayers of lecithin, lysolecithin and fatty acid.

Fluorescence microscopy has recently been proven to be an ideal tool to investigate the specific interaction of phospholipase A2 with oriented substrate monolayers. Using a dual labeling technique, it could be shown that phospholipase A2 can specifically attack and hydrolyze solid analogous L-alpha-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 monolayer is necessary for the observed aggregation of phospholipase A2, mixed monolayers of D- and L-alpha-DPPC, L-alpha-lysoPPC and palmitic acid in different ratios were examined. The phase behavior and the interaction of these films with phospholipase A2 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 A2 underneath these mixed domains results in rapid binding of the protein to the domains without visible hydrolytic activity, regardless of whether the L-form or 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 A2 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 A2. In addition, however, small amounts of D- or L-alpha-DPPC headgroups within the domains of the monolayer seem 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-alpha-DPPC. The acid--immiscible with lecithin--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.

Interaction of bombolitin III with phospholipid monolayers and liposomes and effect on the activity of phospholipase A2.

This study is focused on the characterization of the interaction of the amphiphilic peptide bombolitin III (from the bumblebee Megabombus pennsylvanicus) with phospholipid monolayers and vesicles. It is shown that due to the amphiphilic character of its alpha-helical conformation this water-soluble peptide is able to interact in an ordered fashion with phospholipid organized structures. Depending on the temperature, the subphase, and the particular phosphatidylcholine used, the mixed peptide-phospholipid monolayers can be homogeneous or display phase separation. This behavior was observed by means of the Langmuir film balance technique, coupled with an epifluorescence microscope. In well-defined conditions it is possible to visualize the formation of phase-separated peptide domains at the air-water interface and to study the effect of their presence on the organization of the lipid. The action of phospholipase A2 at the lipid-peptide interface was also followed by means of fluorescence microscopy: some evidence that the enzyme preferentially hydrolyzes the phospholipid that is in contact with the peptide is presented. Furthermore, the presence of bombolitin III in L-alpha-DLPC monolayers causes an increase in the initial speed of degradation with phospholipase A2. These results are in agreement with previous findings that show that the bombolitins are activators in vitro of phospholipase A2. Experiments were also performed with peptide fragments corresponding to the alpha-helical sequences of the protein uteroglobin: despite some amphiphilic character, these peptides do not interact strongly with phospholipid monolayers. Only one of these peptides (corresponding to the helix 4-14 in uteroglobin) is adsorbed in the monolayer in a similar fashion to bombolitin III but does not cause an increase in the activity of phospholipase A2.