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.     

Nonsolvent water in liposomes

Summary In liposomes of dimyristoyl lecithin at 40°C, a quantity of water equal to about 11.5 moles water per mole lecithin, or about one-third of the enclosed liposome water or one-fifth of the total pellet water, behaves as if it is unavailable for dissolving sucrose. This phenomenon represents permanent exclusion of sucrose, not simply a space that equilibrates slowly due to the low permeability of sucrose. The amount of nonsolvent water increases with temperature, and is similar to the amount of water bound to the phosphorylcholine groups as estimated by other methods. Nonsolvent water arises from a combination of the forces responsible for salting-out of nonelectrolytes from aqueous solutions by ions, and of steric effects adjacent to a surface. Measured liposome: water partition coeffecients must be corrected for the effect of nonsolvent water.     

The role of hydrogen bonding via interfacial water molecules in antigen-antibody complexation. The HyHEL-10-HEL interaction

To study the role of hydrogen bonding via interfacial water molecules in protein-protein interactions, we examined the interaction between hen egg white lysozyme (HEL) and its HyHEL-10 variable domain fragment (Fv) antibody. We constructed three antibody mutants (l-Y50F, l-S91A, and l-S93A) and investigated the interactions between the mutant Fvs and HEL. Isothermal titration calorimetry indicated that the mutations significantly decreased the negative enthalpy change (8-25 kJ mol(-1)), despite some offset by a favorable entropy change. X-ray crystallography demonstrated that the complexes had nearly identical structures, including the positions of the interfacial water molecules. Taken together, the isothermal titration calorimetric and x-ray crystallographic results indicate that hydrogen bonding via interfacial water enthalpically contributes to the Fv-HEL interaction despite the partial offset because of entropy loss, suggesting that hydrogen bonding stiffens the antigen-antibody complex.     

The role of hepatocytes in the clearance of liposomes from the blood circulation

In this chapter we summarize literature and describe in more detail our own observations over a period of nearly two decennia on the role of hepatocytes in the hepatic clearance of intravenously administered liposomes. Evidence is presented indicating that, although size is an important parameter, it is not decisive in determining access of liposomes to the hepatocytes. Also lipid composition is an important parameter, including charge, rigidity and headgroup composition. The role of the fenestrated sinusoidal endothelial cells in determining liposome accessibility of hepatocytes is discussed as well as the involvement of opsonizing plasma proteins such as apolipoprotein E. Our observations led us to postulate the existence of at least four different mechanisms of interaction of liposomes with hepatocytes, i.e. an endocytic and a non-endocytic one for both neutral and negatively charged vesicles     

The role of tubulin in the mitochondrial metabolism and arrangement in muscle cells

Tubulin, a well-known component of the microtubule in the cytoskeleton, has an important role in the transport and positioning of mitochondria in a cell type dependent manner. This review describes different functional interactions of tubulin with cellular protein complexes and its functional interaction with the mitochondrial outer membrane. Tubulin is present in oxidative as well as glycolytic type muscle cells, but the kinetics of the in vivo regulation of mitochondrial respiration in these muscle types is drastically different. The interaction between VDAC and tubulin is probably influenced by such factors as isoformic patterns of VDAC and tubulin, post-translational modifications of tubulin and phosphorylation of VDAC. Important factor of the selective permeability of VDAC is the mitochondrial creatine kinase pathway which is present in oxidative cells, but is inactive or missing in glycolytic muscle and cancer cells. As the tubulin-VDAC interaction reduces the permeability of the channel by adenine nucleotides, energy transfer can then take place effectively only through the mitochondrial creatine kinase/phosphocreatine pathway. Therefore, closure of VDAC by tubulin may be one of the reasons of apoptosis in cells without the creatine kinase pathway. An important question in tubulin regulated interactions is whether other proteins are interacting with tubulin. The functional interaction may be direct, through other proteins like plectins, or influenced by simultaneous interaction of other complexes with VDAC.     

The role of glucolipid in the biosynthesis of glycoprotein in Tetrahymena pyriformis

Cell-free preparations from Tetrahymena pyriformis catalyze the incorporation of glucose from UDP-glucose into a glucolipid having properties which are identical to those of other dolichyl phosphoryl sugar derivatives. Kinetic and other experiments have provided evidence that this glucolipid serves as glucose donor for two other types of glucosylated substances, one of which has been tentatively identified as an oligosaccharide lipid and the other a glycoprotein or glycoproteins. In addition, the partially purified glucolipid served as a glucosyl donor to these cell components, suggesting that in this protozoan, at least part of the glycoprotein is synthesized by reactions involving lipid-linked sugars in a manner analogous to that which has been observed in glycoprotein synthesis in mammalian cells.     

The role of N-linked oligosaccharides in glycoprotein function.

N-linked glycoproteins include such biologically important molecules as cell-surface receptors, cell-adhesion molecules, immunoglobulins and other serum proteins, and tumor antigens. Investigating the role of carbohydrate in glycoprotein function has included the use of glycosylation inhibitors or site-directed mutagenesis of specific glycosylation sites to prevent the addition of carbohydrate, or glycosylation processing inhibitors or animal cell glycosylation mutants to alter carbohydrate structure. In some proteins, glycosylation plays an important role in recognition, while in others, it may stabilize and/or control the conformation of the protein. The cloning of genes in bacteria or lower eukaryotes--with the goal of producing biologically active proteins for biotechnological purposes--necessitates a better understanding of the role of specific carbohydrate structures.     

Interaction of the glycoprotein from the Bacillus pumilis cell wall with liposomes

The methods of centrifugation and gel-filtration on Sephadexes G-50 and G-150 were used to study the interaction of Bacillus pumilis cell wall glycoprotein component having the molecular weight of 50 kDa (GP-50) with lyposomes from bacterial lipids. GP-50 is shown to sorb on such liposomes and disturb their barrier properties inducing yield of low-molecular label. GP-50 exerts no effect on properties of liposomes from egg lecithin. Electrostatic forces are supposed to play a decisive role in initial acts of GP-50 interactions with lipid phase of microbial envelopes.     

The role of zooplankton on the self-purification in water bodies

Summary The processes of self-purification in polluted waters can be carried out in two ways. First by the conversion of unstable organic matter into the bodies of living organisms. Second the mineralization of organic substances in the process of destruction of organic matter. In the present article there is shown by a number of examples the possible role of zooplankton in the production and destruction of organic matter in polluted water bodies, their participation in the matter and energy cycle, and also in lowering BOD. It has been shown that the production of crustacean zooplankton can reach great amount up to 50 kg/m³ wet weight. The role of Rotatoria in the destruction of organic matter can be 1–17% of the total amount of it in the mass of water over 24 hours, and the role of Cladocera in its mass development approaches 100%. Coefficient of the utilization of food on growth for Cladocera in summer months = 0.33–0.35, for Copepoda = 0.22. The coefficient of the utilization of assimilated food on growth K₂ = 0.41–0.45 for all species. It is noted that with the mass development of zooplankton, its direct participation in the lowering of BOD₁ reaches very high values. So for a biomass of 500 mg/l it is about 40 mg/l O₂ for 24 hours; of B = 3000 mg/l it is 300 mg/l O₂.Thus under conditions of mass development the role of zooplankton in the processes of self-purification of polluted water bodies can be very significant.

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Zusammenfassung Die Prozesse der Selbstreinigung in verunreinigten Gewässern können in zwei Wegen verlaufen. Erstens handelt es sich um die Umwandlung der nicht stabilen organischen Stoffe in die Körper der lebenden Organismen, zweitens um die Mineralisation im Prozesse der Destruktion der organischen Stoffe.Im vorliegenden Beitrag ist die mögliche Rolle des Zooplanktons an der Produktion and Destruktion der organischen Stoffe in verunreinigten Gewässern, sein Anteil am Stof und Energiekreislauf und an der Erniedrigung des BSB in mehreren Beispielen gezeigt. Es wurde gefunden, daß die Produktion der planktischen Krustazeen bis 50 kg/m³ (Frischgewicht) erreichen kann. Der Anteil der Rädertiere an der Destruktion der organischen Stoffe kann im Totalvolumen des Wassers innerhalb von 24 Stunden 1 bis 17% sein, während sich die Vertretung der Kladozeren in ihrer Massenentwicklung bis zu 100% nähert. Das Koeffizient der Ausnutzung der assimilierten Nahrung fur das Wachstum K₂ gleicht bei allen Arten 0,41 bis 0,45. Es wurde beobachtet, daß mit der Massenentwicklung des Zooplanktons seine direkte Beteiligung an der Erniederigung des BSB₂ sehr hohe Werte erreichte, z.B. für eine Biomasse von 500 mg/l Frischgewicht sind es etwa 40 Mg/1 O₂.24 St., für 3000 mg/l etwa 300 mg/l.Es konnte bewiesen werden, daß wahrend einer Massenentwicklung die Rolle des Zooplanktons bei der Selbstreinigung in verunreinigten Gewässern ein bedeutsames Ausmass erreichen kann.

     

Cooperativity in Scapharca dimeric hemoglobin: simulation of binding intermediates and elucidation of the role of interfacial water

Cooperative binding of ligands to proteins can serve to increase their efficiency and to regulate their activity. Thus, understanding of the mechanism of cooperativity is one of the central concerns of molecular biology. For the tetrameric human hemoglobin (HbA), the cooperative mechanism involves a reasonably well understood combination of tertiary and quaternary changes that occur during the binding process. The dimeric hemoglobin of Scapharca (HbI), which is composed of subunits with the same fold as in HbA, is also highly cooperative but the structural changes on ligand binding are small. A re-orientation of Phe97 in the binding pocket and changes in the number of interfacial water molecules have been implicated in the cooperative mechanism. To explore the role of these factors, we have investigated models of partially liganded intermediate states of HbI with molecular dynamics simulation methods. Since, unlike HbA, no structures for intermediates are available, they were constructed by combining subunits from the unliganded and liganded dimers. Two structurally distinct intermediates were examined, and it was shown that the transition between the two intermediates is directly coupled to the number of interfacial water molecules. Further, it was found that there is a well-defined water channel that connects the interface between the subunits to bulk water. The bottleneck (gate) of the channel, which can be open or closed, is made of hydrophilic residues. The implication of the present results for the cooperative mechanism of HbI is discussed.     

Effect of glycerol on the interfacial properties of dipalmitoylphosphatidylcholine liposomes as measured with merocyanine 540.

Liposomes of dipalmitoylphosphatidylcholine (DPPC) prepared in increasing glycerol/glucose ratios show an increase in the absorbance at 570 nm of merocyanine spectra at temperatures below the phase transition. Since this effect is not observed when liposomes are prepared in solutions containing solely glucose, it is attributed to specific interactions of glycerol with the membrane phase. The increase in the 570 nm absorbance is ascribed to a partial fluidification of the membrane interface and is dependent on the distribution of the dye between the inner and the outer compartments of the liposomes and on their osmotic state. The greatest differences in the absorbance ratio are obtained when merocyanine is added to the external media. In consequence, the changes in the spectra of MC are dependent on the surface state of the liposomes which can be modified by an increase of glycerol or glucose in the external media. The present results are examined in the light of the perturbations that glycerol can induce on the barrier properties of the bilayer.     

Selective compartmentation of the hydration products of carbon dioxide in liposomes,and its role in regulating water movement

Using liposomes, some possible consequences of auxin-stimulated proton secretion in plant tissues are modelled. Liposomes, made specifically permeable to protons and potassium ions, but containing at least one impermeant, swell at a rate which is proportional to the availability and hydration rate of carbon dioxide; the gas functions as a source of proton and dehydrated anion and this enables a net transfer of KHCO₃ to take place.     

Role of liposomes in the presentation of HIV envelope glycoprotein and the immune response in mice

The role of antigen presentation in the induction of humoral as well as cell-mediated immune responses has been investigated by anchoring HIV-1 envelope glycoprotein (gp 160/120) into the phospholipidic bilayer of preformed liposomes to produce HIV-Immunosomes. HIV-Immunosomes induced high titres of HIV-specific antibodies when tested by ELISA, IFA and neutralization, whereas equal amounts of purified glycoprotein alone produced lower antibody response. Similarly, HIV-Immunosomes induced antigen-specific Interleukin-2 production and blastogenic response upon restimulation with the same antigen, in animals vaccinated with HIV-Immunosomes, whereas no secondary response was observed in animals vaccinated with equal amounts of purified gp 160/120. Taken together, these results underline the importance of antigen presentation in the establishment of an adequate immune status and show the potential of HIV-Immunosomes as vaccine against AIDS.     

Selective compartmentation of the hydration products of carbon dioxide in liposomes, and its role in regulating water movement.

Using liposomes, some possible consequences of auxin-stimulated proton secretion in plant tissues are modelled. Liposomes, made specifically permeable to protons and potassium ions, but containing at least one impermeant, swell at a rate which is proportional to the availability and hydration rate of carbon dioxide; the gas functions as a source of proton and dehydrated anion and this enables a net transfer of KHCO3 to take place.     

Fusion of Newcastle disease virus with liposomes: role of the lipid composition of liposomes

We demonstrate here that fusion occurs between the membrane of the Newcastle disease virus (NDV) and liposomes. Fluorescence dequenching studies (using Rhodamine-bearing viral envelopes) revealed the mixing of the lipids constituting the viral and liposomal membrane. The digestion of internal viral proteins by trypsin-containing liposomes indicated the mixing of the internal aqueous compartments. This last assay is independent of exchange of lipids between liposomal and viral membrane in the absence of fusion. Investigation of the effects of liposomal composition indicated that the presence of phosphatidylethanolamine and gangliosides are essential to optimize fusion. The fact that the Newcastle disease virus membrane can fuse with liposome also confirms that fusion must be determined by the viral proteins and could be mostly independent of the nature or presence of the host proteins.