Gertrud Puu

From Liposomes to Supported, Planar Bilayer Structures on Hydrophilic and Hydrophobic Surfaces: An Atomic Force Microscopy Study

The sequence of events involved in the transition from attached liposomes to bilayer patches on hydrophilic and hydrophobic solid supports were visualized in situ by Tapping Mode atomic force microscopy in liquid. In a smooth manner, the attached liposomes spread and flattened from the outer edges toward the center until the two membrane bilayers were stacked on top of each other. The top bilayer then either rolls or slides over the bottom bilayer, and the adjacent edges join to form a larger membrane patch. This is clearly visible from the apparent height of 6.0-7.5 nm of the single bilayer, measured in situ. The addition of calcium appeared to increase the rate of the processes preventing the visualization of the intermediate stages. The same intermediate steps appeared to be present on hydrophobic surfaces, although the attached liposomes seemed to be distorted and the resultant membrane edges were uneven. This work has provided visual and detailed information on liposome coalescence (fusion) onto solid supports and demonstrated how the atomic force microscope can be used to study the process.

PLANAR LIPID BILAYERS ON SOLID SUPPORTS FROM LIPOSOMES : FACTORS OF IMPORTANCE FOR KINETICS AND STABILITY

One method to create planar lipid bilayers on solid substrates involves the transfer of lipids from liposomes to the support. We have varied the composition of liposomes systematically using factorial experimental designs and analyzed the adsorption behaviour of lipids from these liposomes onto solid supports. The hydrophilic supports were either used plain or modified with a monolayer of a lipid mixture, exposing hydrophobic groups. The monolayer-covered supports were used to identify factors important for adhesion and stability. Lipid adsorption kinetics was primarily studied on plain silicon supports in an ellipsometric cell or on a silicon nitride surface in a resonant mirror system (IAsys), using the systematic approach. Saturated phospholipids were essential for the required stability. Mixtures of dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, dipalmitoylphosphatidylethanolamine and cholesterol in combination with proteins were investigated in further detail as regards kinetics. The propensity to form a supported planar bilayer could be manipulated by the presence of calcium ions.

Reactivation of nerve agent inhibited human acetylcholinesterases by HI-6 and obidoxime

Acetylcholinesterase was purified from human caudate nucleus and skeletal muscle. The enzyme preparations were used to study aging and reactivation by HI-6 and obidoxime after inhibition by soman and its isomers. HI-6 was found to be the most potent reactivator. For both enzyme preparations a higher reactivatability and a higher rate of aging were observed after inhibition by C+-soman than after inhibition by C(-)-soman. Aging was retarded by propidium diiodide. Reactivation by the two oximes was also studied after inhibition by tabun, sarin and VX. Tissue homogenates were used for this part of the work. Our conclusion is that HI-6 is superior to obidoxime for human acetylcholinesterases inhibited by soman and sarin, while obidoxime is better towards tabun-inhibited enzyme.

Liposome and proteoliposome fusion onto solid substrates, studied using atomic force microscopy, quartz crystal microbalance and surface plasmon resonance. Biological activities of incorporated components

Stable lipid monolayers were deposited using the Langmuir-Blodgett technique onto silicon wafers, and were fused with liposomes containing acetylcholinesterase. The activity of the protein on the solid substrate was verified up to 28 days with a half-life of about 7 days. Atomic force imaging showed that the topography of the surface did not change significantly during this period. Liposomes containing the ganglioside GM1 were fused onto modified gold surfaces using the same technique. The fusion process and binding of cholera toxin to the ganglioside were continously monitored with a quartz crystal microbalance and by surface plasmon resonance. The fusion process resulted in an environment in which biological components retain their activity and, in combination with either one of the techniques used, may be the principle for membrane-based biosensors.

Retained activities of some membrane proteins in stable lipid bilayers on a solid support

Highly stable lipid bilayers, composed of biologically relevant lipids such as phosphatidylcholine, phosphatidylethanolamine and cholesterol, were formed on platinum surfaces. Bacteriorhodopsin isolated from purple membrane (PM) from Halobacterium halobium, cytochrome oxidase from bovine heart, acetylcholinesterase from bovine brain and the nicotinic acetylcholine receptor from Torpedo electric organ were also incorporated into these reconstituted membranes. The proteins retained their biological activities. Some of them were active several weeks after the reconstitution and after several cycles of assay, washing and storage.

Distribution and stability of membrane proteins in lipid membranes on solid supports

Bacteriorhodopsin and the nicotinic acetylcholine receptor were biotinylated and reconstituted in lipidic membranes on silicon supports by fusion with proteoliposomes. The presence and distribution of the proteins were studied by binding with streptavidin. Radio-labelled streptavidin was employed for quantifying the amounts of protein remaining in the supported membranes after storage in buffer. The proteins within the membranes remained bound to the surface for weeks. The biological activity of reconstituted unlabelled receptor upon storage showed stability in membranes formed on silicon supports and a reduced stability when formed onto lipid monolayer covered supports. Atomic force microscopy studies on preparations in liquid showed bilayer structures but also attached, partly fused liposomes and membrane particles. In air, the surface was smoother and contained less of liposomes and more of stacked lipid layers. Preparations labelled with streptavidin conjugated to colloidal gold and imaged in air showed the proteins individually distributed, with no protein-rich patches or protein aggregates.

Stereoselectivity of enzymes involved in toxicity and detoxification of soman

The fate of the four stereoisomers of soman [0-(1,2,2-trimethylpropyl)-methyl-fluoro phosphonate] has been studied a) in vivo in mouse blood and liver after IP injection of 0.75 × LD50 Rc- and Sc-soman respectively, and b) in vitro upon incubation wih acetyl- und pseudocholinesterase, aliesterase and phosphorylphosphatase. The analytical method used is based on gas chromatography — mass spectrometry with deuterated internal standard.Most soman disappeared very rapidly from blood and liver. In liver, SCRP and RCRP, the two isomers that preferentially react with cholinesterase, could be detected. The level of SCRP, which was higher than that of RCRP, could be followed for 17–18 h. In blood only SCRP could be detected. The amounts found were fairly constant during the time period 2 min to 4h, and it could even be detected 17–18 h after soman administration.

Helicobacter pylori interactions with human gastric mucin studied with a resonant mirror biosensor.

Helicobacter pylori, a human pathogen colonizing the gastrointestinal tract, is first interacting with mucus glycoproteins to penetrate the gastric mucus layer and then attach to specific epithelial cell targets. An optical biosensor technique based on the resonant mirror was used to study H. pylori interactions with human gastric mucin. The mucin preparation was immobilized on the sensor surface to set up the experimental model, close to the in vivo situation. Both sialylated and sulphated oligosaccharides interfered with the H. pylori binding to the immobilized mucin by reducing or abolishing the binding of the bacteria. Furthermore, the displacement of the bacteria from immobilized mucin by highly sulphated glycosaminoglycans was observed.

Liposome and phospholipid adsorption on a platinum surface studied in a flow cell designed for simultaneous quartz crystal microbalance and ellipsometry measurements

Abstract A flow cell was designed for simultaneous mass measurements with a quartz crystal microbalance and ellipsometry and was used for studying the adsorption processes of different liposomes, prepared from phospholipids and cholesterol, on both bare and lipid monolayer-covered platinum surfaces. Ellipsometric and gravimetric data were collected during recirculation of liposomes in 0.15 M NaCl. Experiments with ATP-containing liposomes with subsequent lysis were also performed. The results are discussed in terms of both intact liposome adhesion and intact liposome adhesion in combination with planar bilayer formation.

Comparison of kinetic parameters for acetylthiocholine, soman, ketamine and fasciculin towards acetylcholinesterase in liposomes and in solution

Purified acetylcholinesterase from bovine brain was reconstituted by a detergent depletion technique into liposomes, prepared from soybean lecithin. The kinetics for the substrate acetylthiocholine and for three inhibitors with very different binding properties was studied. The results were compared with results from corresponding experiments with solubilized enzyme in detergent solution. The reconstituted enzyme showed a higher affinity for acetylthiocholine, ketamine and fasciculin. Parameters unaffected by the reconstitution were: turnover number for the substrate; the non-competitive component in ketamine inhibition and the kinetics for the active site-directed irreversible inhibitor soman.