melitten and pyrene

The excimer forming capacity of the fluorescent moiety pyrene is employed to measure continuously the transbilayer (re)distribution of a pyrene-labeled phosphatidylcholine analogue (pyPC) in liposomal membranes. pyPC with a lauroyl residue (sn-1 position) and a short (butyroyl) fatty acid chain (sn-2 position) bearing the pyrene moiety incorporates rapidly into the outer leaflet of liposomes. The fluorescence intensities of excimers (I(E)) and of monomers (I(M)) of pyPC depend on the concentration of the analogue in a membrane leaflet. Therefore, the redistribution of pyPC from the outer to the inner leaflet can be followed by changes of the ratio I(E)/I(M). The transbilayer movement of pyPC in pure phospholipid vesicles is very slow indicated by a constant I(E)/I(M). However, addition of membrane active peptides (melittin, magainin 2 amide or a mutant of magainin 2 amide) induced a rapid translocation of pyPC from the outer to the inner leaflet. An approach is presented which allows estimating the transbilayer distribution of pyPC from the measured ratio I(E)/I(M).

Topics: Antimicrobial Cationic Peptides; Buffers; Lipid Bilayers; Magainins; Melitten; Peptides; Phospholipids; Pyrenes; Spectrometry, Fluorescence; Xenopus Proteins

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

The effects of bee venom melittin on the order and dynamics of dimyristoylphosphatidylcholine unilamellar and multilamellar vesicles at a protein-to-lipid molar ratio of 1:60 have been investigated by employing the techniques of nanosecond emission anisotropy with 1,6-diphenyl-1,3,5-hexatriene as the fluorescent probe, enhancement by polar groups of the weakly allowed 0-0 vibronic transition in the fluorescence spectrum of pyrene, and Raman spectroscopy. The emission anisotropy results, which are found to be consistent with the wobble-in-cone model, show that the protein induces an increase in the order parameter, S, of the acyl chains of unilamellar vesicles below, at, and above their phase transition temperature, Tt, and it decreases strongly the diffusion rate, Dw, only below Tt. On the other hand, for multilamellar vesicles, the protein induces a decrease in S only at Tt and does not affect Dw. These effects are consistent with the observed changes in the degree of enhancement of the 0-0 vibronic transition of pyrene. Moreover, the protein broadens the thermal transition profile of multilamellar vesicles but sharpens dramatically that of unilamellar vesicles and fuses them without changing significantly the Tt in either case. On the other hand, the Raman data detect a decrease in the inter- and intramolecular order of the acyl chains of multilamellar vesicles below Tt and a decrease of only the former Tt. This disparity between the Raman and the nanosecond emission anisotropy data is discussed in terms of differences in the time scales of the two techniques and in the state of aggregation of the lipid-bound melittin. The data for the enhancement of the 0-0 vibronic transition of pyrene suggest that, for a melittin-to-lipid ratio of 1:60, the size or structure of channels formed in the bilayer by melittin does not allow the penetration of a neutral molecule the size of pyrene deeply into the bilayer.

Topics: Bee Venoms; Dimyristoylphosphatidylcholine; Diphenylhexatriene; Lipid Bilayers; Liposomes; Melitten; Membrane Fluidity; Membrane Fusion; Pyrenes; Spectrometry, Fluorescence; Spectrum Analysis, Raman