J. Leunissen-Bijvelt

Divalent cations and chlorpromazine can induce non-bilayer structures in phosphatidic acid-containing model membranes.

(1) The structural organization of aqueous dispersions of 1,2-dioleoylphosphatidic acid has been investigated by freeze-fracture electron microscopy in relation to variations in pH, divalent cations and the local anaesthetic chlorpromazine. (2) In the pH range 4--8 in the presence of 100 mM Na+, dioleoylphosphatidic acid is organized in bilayers. (3) At pH 6 and not at pH 4 and 8.5 addition of Ca2+, Mg2+, Mn2+ and chlorpromazine results in the formation of the hexagonal HII phase. (4) Ca2+ and chlorpromazine addition to mixed phosphatidylcholine-dioleoylphosphatidic acid bilayers at pH 6 results in the formation of lipidic particles.

Gramicidin promotes formation of the hexagonal HII phase in aqueous dispersions of phosphatidylethanolamine and phosphatidylcholine

Abstract It is shown by 31P-NMR and electron microscopy that gramicidin promotes the formation of the hexogonal HII phase in aqueous dispersions of dielaidoylphosphatidylethanolamine and dioleoylphosphatidylethanolamine, when present in molar ratios of 1 : 200 and higher. In addition gramicidin also induces the hexogonal HII phase in aqueous dispersions of dioleoylphosphatidylcholine, when present in molar ratios of 1 : 25 and higher.

Gramicidin induces the formation of non-bilayer structures in phosphatidylcholine dispersions in a fatty acid chain length dependent way

The hydrophobic peptide gramicidin is shown by 31P-NMR, freeze-fracture electron microscopy and small-angle X-ray diffraction, to induce a hexogonal HII-phase lipid organization when incorporated in liquid crystalline saturated and unsaturated synthetic and natural phosphatidylcholines if the length of the fatty acids exceeds a 16 carbon atoms chain. The amount of hexagonally organized lipid increases with increasing fatty acid chain length. With phosphatidylcholines possessing shorter fatty acid chains, as well as with the longer phosphatidylcholines in the gel state, a lamellar organization results. Of the various possible models to explain the induction of the hexagonal HII phase by gramicidin, one in which gramicidin dimers span adjacent cylinders of the hexagonal HII phase seems most plausible. In phosphatidylcholines with intermediary chain lengths gramicidin induces intermediary structures, such as lipidic particles and possibly cubic phases. These experiments suggest that the balance between the length of the hydrophobic domain of a peptide and the membrane thickness is of critical importance for the structure of the membrane. In relation to this observation, the possible involvement of non-bilayer lipid structures in insertion and anchoring of membrane proteins is discussed.

Interaction of melittin with negatively charged phospholipids: Consequences for lipid organization

A characterization of the structural alterations induced by melittin in model‐membranes of dioleoylphosphatidic acid and egg phosphatidylglycerol is presented, based on the use of 31P‐NMR, freeze‐fracture electron microscopy and small angle X‐ray scattering. In accordance with earlier findings on the cardiolipinmelittin system, melittin is found to have an inverted phase inducing effect on these negatively charged lipids, in contrast to the influence on zwitterionic phospholipids. In phosphatidic acid this is expressed in the formation of an HII phase; in phosphatidylglycerol a less ordered, non‐lamellar structure with low water content is adopted.

Ultrastructural changes of sarcolemma and mitochondria in the isolated rabbit heart during ischemia and reperfusion

Isolated rabbit hearts were perfused by the Langendorff technique, made ischemic and subsequently reperfused. It was found that ischemia results in: (i) aggregation of the intramembranous particles in the sarcolemma and (ii) extrusion of pure lipidic multilamellar structures (liposomes) from swollen mitochondria. Subsequent reperfusion resulted in further aggregation of the sarcolemmal intramembranous particles and disruption of the sarcolemma, which was attended by the formation of liposome-like structures. Intramembrane particle aggregation is explained in terms of lateral phase separation of the membrane lipids and a reduction of repulsive forces between the membrane proteins, both induced by a decrease in pH and an increase in Ca2+ concentration intracellularly. The formation and extrusion of the multilamellar structures are discussed in terms of destabilization of the bilayer which results in a structural blebbing-off of pure lipid.

Mixtures of gramicidin and lysophosphatidylcholine from lamellar structures

Abstract It is shown by 31P-NMR and freeze-fracture electron microscopy that in aqueous dispersions of mixtures of gramicidin and palmitoyllysophosphatidylcholine lamellar structures are formed which contain four lysophosphatidylcholine molecules per gramicidin monomer.

Ca2+-induced changes in the barrier properties of cardiolipin/phosphatidylcholine bilayers

(1) A selective increase in permeability is induced in cardiolipin/phosphatidylcholine bilayers at Ca2+ concentrations of 1--3 mM. At higher concentrations of Ca2+ the permeability barrier is completely destroyed. (2) The selective increase in permeability is correlated with the formation of lipid particles visualized by freeze-fracture electron microscopy and an isotropic signal in 31P-NMR spectra. (3) Lowering the Ca2+ concentration shows reduction in permeability but the formation of the lipid particles is a non-reversible process. (4) At higher Ca2+ concentrations, 31P-NMR spectra and freeze-fracture results indicate the formation of the hexagonal phase, explaining the disappearance of the permeability barrier.

Steric hindrance in immunolabelling

In this paper we describe immunocytochemical detection of PhoE pore protein in the outer membrane of E. coli K‐12 cells in dependence of a variety of labelling approaches.

Accumulation of LamB-LacZ Hybrid Proteins in Intracytoplasmic Membrane-like Structures in Escherichia coli K12

The subcellular location of LamB-LacZ hybrid proteins in the Escherichia coli K12 strains pop3234 and pop3299 was investigated by immunocytochemical detection and protease-accessibility experiments. Induction of the synthesis of the hybrid proteins resulted in the appearance of membrane-like structures within the cytoplasm of the cells. Labelling of ultrathin cryosections of the cells with anti-beta-galactosidase or anti-LamB protein serum and protein-A-gold complexes revealed that the hybrid proteins were associated with these membrane-like structures or accumulated within the cytoplasm. Protease-accessibility experiments confirmed this localization. Moreover, when low quantities of hybrid proteins were produced, i.e. in uninduced pop3234 cells or in induced pop3299 cells, the hybrid proteins were accessible to trypsin from the periplasmic side of the inner membrane, leaving protected fragments with an apparent Mr of 83,000. Apparently, these hybrid proteins are partly translocated through the inner membrane, resulting in membrane-spanning forms of the proteins.

External addition of gramicidin induces the hexagonal HII phase in dioleoylphosphatidylcholine model membranes

Abstract 31 P-NMR, small angle X-ray diffraction and freeze-fracture electron microscopy show that dioleoylphosphatidylcholine liposomes undergo a transition from the lamellar to the hexagonal H II phase upon injection of an ethanolic solution of gramicidin in the aqueous medium, when the molar ratio of peptide to lipid is 1 to 20 or higher.