ABSTRACT
Chemically modified lysozymes, namely: N-succinyl lysozyme, glycine methyl ester of N-succinyl lysozyme and oxoindole lysozyme have been prepared. Aggregation, fusion and leakage of phospholipid vesicles induced by these derivatives have been studied in comparison with the effect of the unmodified protein. The experiments were carried out with negatively charges 9PC/PA, 9:1) and uncharged (PC and PC/DOPE/Chol (10:5:5)) lipid vesicles of different packing. Fusion and aggregation of negatively charged phospholipid vesicles in induced by proteins positively charged at pH 7.0 involving electrostatic interactions, a similar pattern on fusion and aggregation of the least stably packed lipid vesicles points also to hydrophobic forces playing a role in the lipid-protein interaction. A conformational change of the protein involved increasing beta-turns, loops and unordered structure at the expenses of beta-sheet without affecting alpha helix content. The conformational effect is necessary to provoke the effects studied, since one of the derivatives (N-succinyl lysozyme) neither changes conformation nor causes aggregation and fusion of vesicles. However, there is no relationship between lysozyme activity and fusion or aggregation of lipid vesicles that catalytic and fusogenci sites of, indicating lysozyme are topographically different.
Subject(s)
Liposomes/metabolism , Muramidase/pharmacology , Animals , Chemical Phenomena , Chemistry, Physical , Cholesterol/chemistry , Glycine/analogs & derivatives , Glycine/pharmacology , Membrane Fusion/drug effects , Muramidase/chemistry , Permeability/drug effects , Phosphatidic Acids/chemistry , Phosphatidylcholines/chemistry , Phosphatidylethanolamines/chemistry , Protein Conformation , Succinates/pharmacologyABSTRACT
We have previously demonstrated that lysozyme induced fusion of negatively charged phospholipid vesicles and have stressed the importance of electrostatic interactions (Posse, E. et al. (1990) Biochim. Biophys. Acta 1024, 390-394). Using centrifugation and fluorescence polarization techniques, we show, in the present paper that lysozyme interacts with negatively charged liposomes (PC/PA, 9:1), but also with neutral liposomes (pure PC). Moreover, the ionic strength and pH of the media did not modify the protein-liposomes interactions. Such interactions induce the spontaneous release of encapsulated Tb-DPA complex in liposomes. Release and fusion of PC/PA liposomes were observed. As indicated by kinetic studies and substrate curves, fusion and release are two uncoupled processes. Taking these and previous results into account we suggest a hypothetical mechanism where a relationship between aggregation, leakage and fusion of liposomes induced by lysozyme interaction is established.
Subject(s)
Muramidase/chemistry , Phospholipids/chemistry , Water/chemistry , Electrochemistry , Models, Molecular , Terbium/analysisABSTRACT
Lysozyme promotes fusion of negatively charged phospholipid vesicles prepared by ethanolic injection. Vesicle fusion was a leaky process as revealed by the release of encapsulated carboxyfluorescein or Tb-DPA complex. Extensive proteolysis of lysozyme inhibited the fusion process. The fusion process was critically dependent on the medium ionic strength; 100 mM of any salt was sufficient to inhibit totally the fusion activity of the protein. The high efficiency of lysozyme (80% RET) was almost constant in the pH range from 4.0 to 9.0, but it was sharply diminished when the pH of the medium was at the isoelectric point of the protein (pI 11.0). Fusion induced by chemically modified lysozyme, showed that the pH profile changed according to the isoelectric point of the protein derivative. These observations stress the importance of electrostatic interactions in the process of fusion induced by lysozyme.
Subject(s)
Membrane Fusion/drug effects , Muramidase/pharmacology , Phospholipids/metabolism , Cell Membrane/ultrastructure , Kinetics , Lipid Bilayers/metabolismABSTRACT
Native insulin causes fusion of negatively charged liposomes in the pH range from 3.0 to 5.5. In marked contrast, insulin with all three amino groups succinylated did not show fusion ability at any pH. On the other hand, insulin amidated with glycine methyl ester with all six carboxyl groups blocked shifted its activity to higher pH, showing a pH range of activity from 3.0 to 7.4. When the carboxyl groups were recovered by hydrolysis of methoxyl groups from glycine methyl ester-treated insulin, the protein obtained (glycyl-insulin with six free carboxyl groups) behaved as native insulin. A good correlation between the isoelectric point values of insulin and its derivatives and their fusion properties was found.
