Temperature-dependent osmotic permeability in glycoproteion containing liposomes

The osmotic water outflow of large multilamellar liposomes containing ₁-acid glycoprotein was measured at a temperature near the lipid's phase transition temperature. The liposomes were formed from a mixture of DPPC, cholesterol and glycoprotein in molar ratios 100:20:1, by continuous sucrose density gradient centrifugation. These liposomes captured 35% of the radiolabeled glycoprotein. The temperature-dependent experiments showed that near phase transition temperature the initial rate of water outflow increased drastically in comparison with glycoprotein free liposomes incubated in buffer containing glycoprotein. We suggested that eventual a channel mechanism may be involved due to spontaneous incorporation of glycoprotein into the bilayer.     

Osmotic water permeability through liposomes in the presence of α1-acid glycoprotein

¹-acid glycoprotein (orosomucoid) from human blood serum was isolated in pure form and then reconstituted into large multilamellar liposomes, consisting of a binary mixture of hen-egg phosphatidylcholine and cholesterol. These liposomes were found to be osmotically sensitive. The osmotic water permeability of proteoliposomes was determined by light-scattering measurements of the osmotic volume changes after mixing with hyperosmotic solutions of potassium salts and aminoglycoside antibiotics. The initial rate of water outflow was measured as a function of glycoprotein concentration in the mixture for the preparation of proteoliposomes. This can serve as an indication for membrane permeability to the solutes used in these experiments. It was shown that aminoglycoside antibiotics passed much faster across the membrane than potassium salts, in the presence of glycoprotein in the liposomes. A recognition pattern in the osmotic behavior of these proteoliposomes was assumed.     

X-Ray studies of the ovomucoid-DHPC system

Complex of ovomucoid with 1,2 dihehadecyl-snglycero-3-phosphatidylcholine (DHPC) in water solution has been used as a model system for glycoprotein-lipid interactions.The structural parameters of this complex were determined with small angle-X-ray diffraction techniques. Knowing the repeat distance, the chemical composition of the association and the partial specific volumes of the components, the partial thickness of the glycoprotein, lipid and water layers can be determined and compared with the thickness of the lipid layers observed in pure lipid-water systems and lamellar associations in the absence of glycoprotein. The variation of the structural parameters at room temperature with the concentration of water was determined. Our results showed: the intersheet spacing increases from 112 to 157 Å, the thickness of the hydrocarbon chain layers decreases from 49 to 40Å and the thickness of glycoprotein layer increases from 62 to 100 Å. In this case the glycoprotein-lipid interaction appears to be of weak electrostatic nature and to involve mainly the polar regions of the structure. Fluorescence experiments have also been carried out to confirm the X-ray data.     

Glycoprotein-protein interaction examined by kinetic studies of pyrene transfer

The transfer of pyrene between ₁-acid glycoprotein, acethylcholinesterase and sonicated liposomes was used to monitor glycoprotein-protein interaction on the lipid bilayer. When a density solution of glycoprotein or protein labeled with pyrene was mixed with unlabeled suspension of free-phospholipid liposomes, or suspensions containing the complexes of glycoprotein-lipid, protein-lipid, or glycoprotein-protein-lipid, pyrene excimer fluorescence increased with a half-time of approximately 30–50 msec. Since the increase in excimer fluorescence indicates an increase in the microscope concentrations of pyrene, the observed fluorescence change reflects pyrene transfer. The half-times for the increase in excimer fluorescence were determined in the presence of glycoprotein and protein in the liposomes. On the basis of the determined half-times it was concluded that both, glycoprotein and protein are bound on the lipid bilayer. Our data also suggest that the thickness of the lipid bilayer is significantly changed in this case. The observation suggests strongly that the limiting step in the transfer of pyrene is not the dissociation of pyrene, but the uptake of the pyrene monomers by the lipid phase.     

Effects of the components of Newcastle disease virus on the structural order of lipid assemblies

The membrane M-protein of Newcastle disease virus is localized directly beneath the lipid bilayer. Although this protein is the major constituent of the virus, its structural relationship to the lipid or to the other viral component hemagglutininneuraminidase, the so called HN-glycoprotein, is still unknown. The effects of either M-protein alone or both M-protein and HN-glycoprotein on the lipid assemblies in reconstituted liposomes were determined by differential polarized phase fluorometry, steady-state fluorescence anisotropy and emission lifetime measurements.It is demonstrated that the degree of rotation of fluorophores in reconstituted liposomes is restricted by the molecular packing of lipids in the bilayer and this in turn can be correlated with the structural order of the lipids in the membrane. The experimental results show that the structural order parameters calculated from the fluorescence measurements are strongly influenced by the presence of both M-protein and HN-glycoprotein in the lipid assemblies.     

The role of membrane bound glycoprotein on the permeability of liposomes

The liposome permeability to potassium and methanosulfonate ions was determined in the presence of bound glycoprotein and protein. The permeability changes were registered by light-scattering measurements of the osmotic volume changes of liposome suspension after mixing with solutions containing K+ and MS- ions respectively. The permeability changes varied considerably with the change of glycoprotein-protein molar ratio in the liposomes. It was suggested that topological distribution of both molecules (glycoprotein and protein) in the lipid bilayer would play a substantial role and influence the permeability. It was confirmed from fluorescence measurements with ANS as a fluorescence marker. There was an increase of the number of binding sites (n) for ANS, increasing glycoprotein-protein molar ratio in the liposomes. These results were interpreted in terms of electrostatical changes of the membrane lipid region and membrane surface, caused by the interaction of glycoprotein and protein with lipids, as well as the associated role of these components on the permeability.     

Osmotic water permeability in glycoprotein containing liposomes

The kinetics of osmotic water permeability in proteoliposomes containing ₁-acid glycoprotein was investigated by means of stopped-flow spectrophotometry. A biphasic time-course of scattered light with time was registered. The rate constants calculated from fits to an exponential function in the first phase were proportional to the final medium osmolarity. The apparent second order rate constants K_app (Osm^-1 sec^-1) were determined at different glycoprotein concentrations in the original mixture for preparation of proteoliposomes. The value of K_app at lipid:glycoprotein weight ratio = 1 was plotted in Arrhenius coordinates. The calculated activation energy for water permeation through the lipid bilayer suggests that eventual channel mechanism may be involved due to the presence of glycoprotein molecule in the liposomes.     

Studies on the kinetic effects of cyclic nucleotides dependent glutamate dehydrogenase

Stopped-flow spectrophotometric studies of the reductive amination of L-ketoglutarate by L-glutamate dehydrogenase showed a biphase time course, which consisted of a rapid first phase lasting 60–100 msec and a slow final phase in which the rate of coenzyme oxidation increased until the coenzyme was depleted. The effects of 3,5-cyclic adenosine monophosphate (cAMP) and 3,5-cyclic guanosine monophosphate (cGMP) on the time course of both phases were established. The results showed that in the concentration ranges used the cyclic nucleotides accelerate the catalytic reaction. The effect of cAMP was more pronounced as compared to cGMP. In all cases this influence was most clearly expressed in the first phase. Using an Arrhenius plot the activation parameters were calculated. The experiments with cAMP and cGMP at different molar ratios showed that a specific cAMP binding may occur.     

Effect of delta-lysin on the phase transitions of lipid assemblies

X-ray small-angle diffraction, differential scanning calorimetry (DSC), and temperature scanning densitometry (TSD) were used to study the effect of -lysin on the phase transitions of lipid assemblies from 1,2-0-dixehadecyl-sn-glycero-3-phosphoholine (DHPC). The experiments were carried out in excess of water in a temperature range of 0–55 °C, and at low peptide concentrations between 10^-4 and 10^-2 moles peptide per mole phospholipid. The incorporation of -lysin into lipid assemblies alters the lipid structure without significant changes on the temperatures of phase transition from gel to liquid crystalline phase. The temperature of the main transition was nearly unaffected. A reduction in the transition volume of the lipids with increasing concentrations of -lysin was observed. The minor changes in these parameters were interpreted as long-range structural changes caused by the peptide incorporation. The results are discussed in terms of the concept of cooperative phase transition of entire clusters occurring within a membrane implying that relative stable domains of gel phase, and liquid crystalline phase co-exist.     

Interaction of albumins from different species with phospholipid liposomes. Multiple binding sites system

The interactions of three serum albumin species (rat, human, and bovine) with liposomes containing dimyristoylphosphatidylcholine, distearoylphosphatidylcholine or mixtures of both under different membrane fluidity conditions have been investigated using isothermal titration calorimetry and steady-state fluorescence anisotropy. Calorimetric titration studies of the binding of liposomes to the albumin species indicate in all cases exothermic processes with multiple sites of binding in the albumin molecules. Distinct saturation of the protein-lipid binding processes was observed at low or high molar lipid/protein ratio depending on the particular system. The thermodynamic parameters, including the association enthalpy and entropy, and the optimal values for the binding constants were thoroughly varied as a function of the number of identical binding sites, defining the best value of the parameter. Our experimental results, obtained using complementary biophysical techniques, provide experimental evidence for a significant difference in the association of the three protein species to phospholipid membranes. These observations also suggest a close relation between the binding parameters of the protein/lipid association and the lipid state of the phospholipid membranes.