1-palmitoyl-2-oleoylphosphatidylcholine and dimyristoylmethylphosphatidic-acid

Intervesicle phospholipid exchange through molecular contacts induced by the C1 molecular species of myelin basic protein (MBP) are characterized by using methods that amplify the effect of MBP-membrane interaction. The effect of salt concentration (KCl) on the vesicle-vesicle interaction of anionic sonicated covesicles of 30% 1-palmitoyl-2-oleoylglycero-sn-3-phosphocholine and 70% 1,2-dimyristoylglycero-sn-3-phosphomethanol (POPC/DMPM) by MBP is dissected by a combination of protocols into individual steps: aggregation of vesicles, apposition and contact formation, and hemifusion. Scattering and resonance energy transfer measurements reveal that, in the absence of KCl, MBP promotes rapid aggregation of the vesicles without lipid mixing. At >40 mM KCl, the extent of aggregation is larger and time-dependent. Fluorescence dequenching due to dilution of labeled phospholipids indicates that on a somewhat slower time scale, hemifusion of vesicles is triggered by salt, with mixing of the outer monolayer lipids but without flip-flop of phospholipids and without mixing or leakage of the aqueous contents. The exchange and hemifusion are seen with anionic vesicles; the effect of the structure of phospholipid, composition of vesicles, and the protein/lipid ratio is primarily on the kinetics of these and other competing processes. Thus, at 0.022 mol % of MBP and less than 100 mM KCl, it is possible to uncouple three sequential steps: (1) aggregation of vesicles by MBP; (2) apposition of bilayers and selective lipid exchange through vesicle-vesicle contacts established by MBP, i.e., anionic and zwitterionic phospholipids exchange, but cationic probes are excluded; and (3) hemifusion and lipid mixing of contacting monolayers of vesicles.

Topics: Anions; Binding Sites; Cations; Energy Transfer; Fluorescent Dyes; Glycerophospholipids; Humans; Infant; Lipid Bilayers; Membrane Fusion; Myelin Basic Protein; Particle Size; Phosphatidic Acids; Phosphatidylcholines; Phospholipids; Potassium Chloride; Rhodamines; Spectrometry, Fluorescence; Water

Mellitin, a cationic amphiphilic peptide, has an apparent activating effect on interfacial catalysis by phospholipase A2 (PLA2) of bee venom on zwitterionic vesicles of 1-palmitoyl-2-oleoylglycero-sn-3-phosphocholine (POPC) and on anionic vesicles of 1,2-dimyristoylglycero-sn-3-phosphomethanol (DMPM), as well as on covesicles of POPC/DMPM (3:7). On the other hand, mellitin-induced increase in the rate of pig pancreatic PLA2 is seen only on anionic vesicles. Interfacial kinetic protocols and spectroscopic methods show that the activation is due to enhanced substrate replenishment resulting from intervesicle exchange of zwitterionic or anionic phospholipids through vesicle-vesicle contacts established by mellitin. It is shown that as the hydrolysis on POPC vesicles progresses, due to a high propensity of bee PLA2 for binding to the product containing zwitterionic vesicles, most of the enzyme in the reaction mixture is trapped on few vesicles that are initially hydrolyzed, and thus reaction ceases. Under these conditions, mellitin promotes substrate replenishment by direct exchange of the products of hydrolysis from the enzyme-containing vesicles with the substrate present in excess vesicles which have not been hydrolyzed. Pig PLA2 has poor affinity for POPC vesicles, and the affinity is only modestly higher in the presence of low mole fractions of the products of hydrolysis; therefore, the enzyme is not trapped on those vesicles. Biophysical studies confirm that the phospholipid exchange occurs through stable intervesicle contacts formed by low mole fractions of mellitin, without transbilayer movement of phospholipids or fusion of vesicles. At high mole fraction (> 1.5%) mellitin induces leakage in POPC vesicles and does not form additional contacts. In POPC/DMPM vesicles, the contacts are formed even at high mole fractions of mellitin. Changes in intrinsic tryptophan fluorescence of mellitin indicate that bound mellitin exists in at least two different functional forms depending on the lipid composition and on the lipid:peptide ratio. A model is proposed to accommodate amphiphilic mellitin as a transmembrane channel or an intervesicle contact.

Topics: Acrylamide; Acrylamides; Amino Acid Sequence; Animals; Bee Venoms; Dithionite; Drug Synergism; Fatty Acids; Fluorescent Dyes; Glycerophospholipids; Hydrolysis; Kinetics; Liposomes; Lysophosphatidylcholines; Melitten; Molecular Sequence Data; Pancreas; Phosphatidic Acids; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipases A; Phospholipases A2; Phospholipids; Pyrenes; Scattering, Radiation; Spectrometry, Fluorescence; Swine