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Biomedical subjects

L I Barsukov

Publications and source records attributed to L I Barsukov.

At least 19 recordsLinked to original sources
PubMed2000

[Synthesis and properties of fluorescent labeled detergents analogues of glycocholic acid].

For studying membrane processes with participation of detergents, fluorescent analogues of glycocholic acid containing p-hydroxybenzyl, 7-nitrobenz-2-oxa-1,3-diazol-4-yl, or fluorescein-5-thiocarbamoyl fluorophore in the glycyl moiety attached to glycocholic acid were synthesized. The fluorophores are in the probes near their carboxyl groups and, in membrane systems, should therefore be situated on the interface and be sensitive to phase transitions. The critical micelle concentrations were determined for the analogues and found to be close to those of cholate and glycocholate in the case of the first two compounds. We presume that the behavior of the probes in membrane systems will mimic the behavior of the bile acid salts.

Detergents↗
PubMed1995

Temperature-induced micellar-lamellar transformation in binary mixtures of saturated phosphatidylcholines with sodium cholate.

The transition states of binary mixtures of dipalmitoyl- and dimyristoylphosphatidylcholines with sodium cholate at the reversible temperature-induced micellar-lamellar transformation were characterized by turbidimetry, electron microscopy, 31P NMR and differential scanning calorimetry. This transformation is triggered by the phospholipid acyl chain melting, and appears to include two structural pathways: (i) from discoidal mixed micelles to network-like structures composed of long interlaced rod-like micelles, then to multilayer membrane structures, and finally to multilamellar vesicles; and (ii) from discoidal micelles to membrane fragments and finally to unilamellar vesicles.

Calorimetry, Differential Scanning↗
PubMed1993

The double pi pi 5.6 helix of gramicidin A predominates in unsaturated lipid membranes.

The structure of the channel-forming polypeptide gramicidin A (GA) incorporated into phosphatidyl-choline (PC) liposomes has been studied as a function of the degree of unsaturation of the acyl chains of PC. The initial conformational state of GA in reconstituted bilayers is determined by the solvent in which the peptide and the lipid are initially co-dissolved, whereas the equilibrium conformational state (after heat incubation) is affected by the lipid structure rather than by the nature of the solvent. The conformational equilibrium of GA has been studied in liposomes prepared from PC having a variable number of double bonds in the fatty acid moiety, by circular dichroism and Fourier transform infrared. Liposomes were prepared from trifluoroethanol or ethanol solutions and incubated at 68 degrees C. GA was shown to retain the conformation of the right-handed pi-->6.3 pi<--6.3 helix in PC with saturated acyl chains and with one double bond, whereas in dilinoleoyl-PC, having two double bond in each chain, the thermodynamically preferred structures are left-handed antiparallel and parallel double pi pi 5.6 helices. Natural soybean PC also favours left-handed pi pi 5.6 helical structures of GA (approximately 75%). This finding is discussed in terms of the role of PC unsaturation in the dynamic properties of the lipid matrix. Differences between observed FTIR spectra of the increases decreases pi pi 5.6 helix in solution (and to a larger extent in the membrane) and the calculated IR spectra can be interpreted as resulting from deviation of the real structure from the theoretically derived ideal helix. The data obtained provide grounds for better understanding of a GA channel functioning in lipids of variable degrees of unsaturation.

Cesium↗
PubMed1991

The divalent cation-binding sites of gramicidin A transmembrane ion-channel.

The conductance of the gramicidin A single channels in glycerolmonooleate membranes is strongly reduced in the presence of Mn2+ cations. The nmr experiments were performed for N-terminal to N-terminal gramicidin A dimer formed by two right-handed single-stranded helixes incorporated into the sodium dodecyl sulfate micelles in the presence of Mn2+ ions. Dependence of the nonselective spin-lattice relaxation rates of the gramicidin A protons on Mn2+ concentration was analyzed to determine coordinates of the divalent cation binding sites. It is inferred that Mn2+ ions are bound at the channel mouths at distances of 6.4, 8.6, and 8.8 A (+/- 2 A) from the oxygen atoms of exposed carbonyl groups of D-Leu 12, 14, and 10, respectively. The bounded Mn2+ retains its hydrate shell, the size of which (approximately 6 A) exceeds the inner pore diameter (approximately 4 A). That makes the gramicidin A channel impermeable for divalent cations.

Binding Sites↗
PubMed1986

Phospholipid topology and flip-flop in intestinal brush-border membrane.

The topological distribution of the two major phospholipids of brush-border membrane, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), has been investigated using brush-border membrane vesicles from rabbit small intestine. Bee venom phospholipase A2 and phosphatidylcholine exchange protein from bovine liver were used as membrane probes. It is shown that the brush-border membrane retains its integrity under conditions of phospholipase hydrolysis and intermembrane phospholipid exchange. Kinetic analysis of the data of phospholipase hydrolysis and phospholipid exchange at temperatures under 10 degrees C shows that both PC and PE occur in two pools: a minor (about 25%) more readily accessible pool and a major one (about 75%) less readily available. The rate of PC exchange between these two pools is relatively fast. The half-time derived under conditions of phospholipase hydrolysis is of the order of 20 min. Under conditions of phospholipid exchange the exchange rates may be even faster. The difference in exchange kinetics observed with the two methods of probing is probably due to changes in membrane properties such as the bilayer fluidity induced by the probing process itself. It is proposed that the two pools represent the transverse distribution of the phospholipids. The two major phospholipids of brush-border membranes, PC and PE, would be distributed mainly on the inner (cytoplasmic) side of the brush-border membrane. The phospholipid exchange between the brush-border vesicles and unilamellar phosphatidylcholine vesicles in the presence of phosphatidylcholine exchange protein reveals that significant quantities of phospholipid are taken up by brush-border membrane independently, i.e., in a separate process independent of the exchange protein-catalyzed phosphatidylcholine exchange.

Androgen-Binding Protein↗
PubMed1982

A sphingomyelin transfer protein in rat tumors and fetal liver.

The binding of the disaccharides methyl beta-D-lactoside and 2-acetamido-2-deoxy-3-O-(beta-D-galactopyranosyl)-beta-D-galactopyranose [beta-D-Gal-(l leads to 3)-D-GalNAc] to peanut agglutinin was studied by ultraviolet difference spectroscopy. The magnitude of the difference spectra varied with the concentration of the carbohydrates; association constants and thermodynamic parameters were determined from titration experiments at different temperatures. The enthalpy and entropy changes for binding of methyl beta-D-lactoside were found to be delta H degree = -65 +/- 4 kJ mol-1, delta S degree = -156 +/- 14 J mol-1 K-1. For beta-D-Gal-(1 leads to 3)-D-GalNAc the observed thermodynamic parameters were delta H degree = -78 +/- 5 kJ mol-1, delta S degree = -177 +/- 16 J mol-1 K-1. For both disaccharides, the enthalpy change upon binding to the lectin is much larger than found for the binding site on peanut agglutinin. The observed parameters are compared with those found for the binding of monosaccharides and oligosaccharides to other lectins and to lysozyme. Molecular models of the minimum energy conformers of beta-D-Gal(1 leads to 3)-D-GalNAc and methyl beta-D-lactoside are used to interpret the interaction of these, and structurally related ligands, with the peanut agglutinin binding site.

Androgen-Binding Protein↗
PubMed1982

[Transmembrane migration of phosphatidylcholine and peroxidation of lipids in rat liver microsomes].

The exchangeability of phosphatidylcholine in microsomes differing in the degree of lipid peroxidation was determined by incubation of microsomes labelled in vivo with [methyl-14C]choline chloride with an excess of phosphatidylcholine liposomes and phosphatidyl-exchange protein from beef liver. The phosphatidylcholine peroxidation products were shown to act as substrates for the phosphatidylcholine-exchange protein. The level of exchangeable microsomal phosphatidylcholine did not depend on the degree of lipid peroxidation in the microsomes. Stimulation of lipid peroxidation by Fe2+-ascorbate or inhibition of lipid peroxidation by alpha-tocopherol did not influence the exchange of microsomal phosphatidylcholine. However, with highly peroxidized phosphatidylcholine liposomes as acceptor particles the exchange of microsomal phosphatidylcholine in the presence of phosphatidylcholine-exchange protein was strongly inhibited. In all cases the percentage of microsomal phosphatidylcholine exchangeable within 1-2 hrs was 85-90%. It is concluded that lipid peroxidation is not responsible for the rapid translocation (flip-flop) of phosphatidylcholine in rat liver microsomes. A possible involvement of membrane proteins in this process is proposed.

Androgen-Binding Protein↗
PubMed1982

[Rapid transmembrane migration of phosphatidylcholine under the influence of cytochrome P-450].

The phosphatidylcholine exchange protein and bee venom phospholipase A2 were used to estimate the phosphatidylcholine accessibility in proteoliposome-containing cytochrome P-450. Without cytochrome P-450 phosphatidylcholine was only partly accessible for the both membrane probes. Incorporation of cytochrome P-450 into the liposomes resulted in a complete accessibility of phosphatidylcholine for the phosphatidylcholine exchange protein and phospholipase A2. On the other hand, phosphatidylcholine was only partly available for the membrane probes in cytochrome b5 containing proteoliposomes. It was concluded that cytochrome P-450 induces rapid transmembrane translocation of phosphatidylcholine with tau 1/2 less than 20 min. It is proposed that cytochrome P-450 may be responsible for rapid flip-flop of phosphatidylcholine in rat liver microsomes.

Androgen-Binding Protein↗
PubMed1980

Investigation of the inside-outside distribution, intermembrane exchange and transbilayer movement of phospholipids in sonicated vesicles by shift reagent NMR.

1. A new NMR approach is described for the investigation of transbilayer asymmetry in phospholipid vesicles consisting of phosphatidylcholine and negatively charged phospholipids. The method makes use of the dependence of the psuedocontact shift of the N-methyl proton resonance induced by paramagnetic ions on the surface concentration of negatively charged phospholipids. When two differently shifting paramagnetic probes are applied from the outside and the inside of a vesicular membrane the transbilayer phospholipid distribution can be estimated without knowledge of the inner and outer radii of the vesicles and the packing density of the phospholipid molecules. 2. The method was employed to study the transbilayer asymmetry in vesicles obtained by cosonication of phosphatidylcholine with phosphatidylserine, phosphatidylglycerol or phosphatidylinositol. The three negative phospholipids were found to distribute with a higher surface concentration in the inner vesicular shell than in the outer one when their total content did not exceed 25 mol%. However, as the amount of negatively charged phospholipids increases the ratio of their inside to outside surface concentrations, i.e., the transbilayer asymmetry of the vesicles, decreases. Prolonged incubation (for several days) does not change the compositional asymmetry of the cosonicated vesicles. 3. By the 'double-probe' technique it was established that spontaneous exchange between separately sonicated phosphatidylcholine and phosphatidylinositol vesicles results in formation of highly asymmetric mixed vesicles with phosphatidylinositol residing only in the outer monolayer. In the presence of antioxidant (alpha-tocopherol) the bilayer asymmetry is preserved for days. However lipid peroxidation induces rapid transbilayer movement (flip-flop) of phospholipids leading to an 'inverted' asymmetry resembling that of cosonicated vesicles. It is suggested that lipid peroxidation promotes phospholipid flip-flop by partially converting the bilayer structure into a non-bilayer configuration. Moderate quantities of lysophosphatidylcholine (up to 15 mol%) induce neither detectable perturbations of the bilayer nor rapid phospholipid flip-flop.

Electrochemistry↗
PubMed1978

Affinity chromatography of the phosphatidylcholine exchange protein from bovine liver.

Affinity chromatography has been used to purify the phosphatidylcholine exchange protein from bovine liver. The affinity resin consisted of 1-acyl-2-(9-carboxy)nonyl-glycero-3-phosphocholine linked to AH-Sepharose 4 B via the carboxyl group. Application of a crude exchange protein fraction to the affinity column resulted in a complete adsorption of the phosphatidylcholine exchange protein. The exchange protein eluted with a buffer containing 0.15% sodium deoxycholate. The most active fraction was 130-fold purified and accounted for 62% of the activity.

Animals↗
PubMed1978

Manipulation of phospholipid composition of membranes with the aid of lipid exchange proteins. Incorporation of phosphatidylcholine into protoplasts of Micrococcus lysodeikticus.

Incubation of Micrococcus lysodeikticus protoplasts with phosphatidylcholine liposomes and rat liver exchange proteins (pH 5.1 supernatant fraction) resulted in replacement of about one half of the bacterial total phospholipids by phosphatidylcholine. Protoplasts modified by phosphatidylcholine showed a decreased rate of oxidation of exogenous substrates (NADH, malate) and decreased ferricyanide reductase activity as compared to the initial protoplasts. At the same time incorporation of phosphatidylcholine had no influence on the level of endogeneous respiration. Protoplasts modified by phosphatidylcholine were osmotically more stable than the initial protoplasts. After osmotic lysis of the phosphatidylcholine protoplasts their NADH (malate) oxidase and ferricyanide reductase activities were restored. Incorporation of phosphatidylcholine into membrane ghosts, obtained by osmotic rupture of the initial protoplasts had only small if any effect on the malate and NADH oxidase and dehydrogenase activities. It is concluded that phosphatidylcholine in incorporated predominantly into the outer part of cytoplasmic membrane and that proteinmediated transfer of phosphatidylcholine results in restoration of the permeability barrier due to repair of local defects in the initial protoplast membrane.

Cell Membrane↗