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Biomedical subjects

Z Salamon

Publications and source records attributed to Z Salamon.

At least 37 records · Page 2Linked to original sources
PubMed1995

Low concentrations of diacylglycerol promote the binding of apolipophorin III to a phospholipid bilayer: a surface plasmon resonance spectroscopy study.

The binding of the exchangeable apolipoprotein apolipophorin III (apoLp-III) to an egg phosphatidylcholine bilayer as a function of the concentration of diacylglycerol (DG) in the bilayer was studied by surface plasmon resonance spectroscopy. At a DG concentration of 2 mol % in the bilayer, the binding of apoLp-III reached saturation. Under saturating conditions, apoLp-III forms a closely packed monolayer approximately 55 A thick, in which each molecule of protein occupies approximately 500 A2 at the membrane surface. These dimensions are consistent with the molecular size of the apoLp-III molecule determined by x-ray crystallography, if apoLp-III binds to the bilayer with the long axis of the apoLp-III normal to the membrane surface. In the absence of protein, the overall structure of the lipid bilayer was not significantly changed up to 2.5 mol% DG. However, at 4 and 6 mol % DG, the presence of nonbilayer structures was observed. The addition of apoLp-III to a membrane containing 6 mol % DG promoted the formation of large lipid-protein complexes. These data support a two-step sequential binding mechanism for binding of apoLp-III to a lipid surface. The first step is a recognition process, consisting of the adsorption of apoLp-III to a nascent hydrophobic defect in the phospholipid bilayer caused by the presence of DG. This recognition process might depend on the presence of a hydrophobic sensor located at one of the ends of the long axis of the apoLp-III molecule but would be consolidated through H-bond and electrostatic interactions. Once primary binding is achieved, subsequent enlargement of the hydrophobic defect in the lipid surface would trigger the unfolding of the apolipoprotein and binding via the amphipathic alpha-helices. This two-step sequential binding mechanism could be a general mechanism for all exchangeable apolipoproteins. A possible physiological role of the ability of apoLp-III to bind to lipid structures in two orientations is also proposed.

Animals↗
PubMed1995

Photobleaching of the photoactive yellow protein from Ectothiorhodospira halophila promotes binding to lipid bilayers: evidence from surface plasmon resonance spectroscopy.

The photoactive yellow protein (PYP) from the phototrophic bacterium Ectothiorhodospira halophila is a small, soluble protein that undergoes reversible photobleaching upon blue light irradiation and may function to mediate the negative phototactic response. Based on previous studies of the effects of solvent viscosity and of aliphatic alcohols on PYP photokinetics, we proposed that photobleaching is concomitant with a protein conformational change that exposes a hydrophobic region on the protein surface. In the present investigation, we have used surface plasmon resonance (SPR) spectroscopy to characterize the binding of PYP to lipid bilayers deposited on a thin silver film. SPR spectra demonstrate that the net negatively charged PYP molecule can bind in a saturable manner to electrically neutral, net positively, and net negatively charged bilayers. Illumination with either blue or white light of a PYP solution, which is in contact with the bilayer, at concentrations below saturation results in an increase in the extent of binding, consistent with exposure of a high affinity hydrophobic surface in the photobleached state, a property that may contribute to its biological function. A value for the thickness of the bound PYP layer (23 A), obtained from theoretical fits to the SPR spectra, is consistent with the structure of the protein determined by x-ray crystallography and indicates that the molecule binds with its long axis parallel to the membrane surface.

Adsorption↗
PubMed1994

Conformational changes in rhodopsin probed by surface plasmon resonance spectroscopy.

Surface plasmon resonance (SPR) spectroscopy has been used to follow incorporation and light-induced conformational changes in bovine rhodopsin reconstituted into an egg phosphatidylcholine bilayer deposited on a thin silver film. The magnitude of the SPR spectral changes caused by light varies with pH in a manner paralleling that in flash photolysis experiments, which monitor formation of metarhodopsin II. Irradiation produces an increase of approximately 4 A in the average thickness of the proteolipid layer, consistent with exposure of recognition sites for the G protein. The results demonstrate that the SPR technology described herein may be used to monitor conformational events in membrane-associated receptors such as rhodopsin.

Animals↗
PubMed1994

Assembly and molecular organization of self-assembled lipid bilayers on solid substrates monitored by surface plasmon resonance spectroscopy.

The structural properties of lipid films, made from a squalene/butanol solution containing varying amounts (0-15 mg/ml) of egg phosphatidylcholine and deposited on a thin metallic silver layer, were investigated using surface plasmon resonance (SPR) spectroscopy. Optical parameters (thickness, refractive index and extinction coefficient) of such supported self-assembled lipid membranes were obtained from a theoretical analysis of the experimental SPR curves. The mass of the lipid membrane and the area and volume occupied by one lipid molecule were also calculated. The results were consistent with the formation of durable and homogeneous lipid bilayers on the solid substrate, and indicated similarities in structural properties between the present lipid bilayers and freely suspended and Langmuir-Blodgett bilayer membranes. Such bilayers represent a simple model for biological membranes, as well as providing a means of immobilizing proteins for various practical applications, including receptor-based sensors and molecular devices. The results confirm the value of the SPR technique for investigating the properties of thin biomolecular dielectric films deposited on a metal surface.

Lipid Bilayers↗
PubMed1994

Transient kinetic and oxidation-reduction studies of spinach ferredoxin:nitrite oxidoreductase.

The oxidation-reduction midpoint potentials for the two prosthetic groups of the chloroplast-located, ferredoxin-dependent nitrite reductase of spinach leaves have been determined by spectroelectrochemical titrations and cyclic voltammetry. The average of the results obtained by the two techniques are Em = -290 mV for the siroheme group and Em = -365 mV for the [4Fe-4S] cluster. The value obtained for the [4Fe-4S] cluster is substantially more positive than values obtained previously in experiments which utilized electron paramagnetic resonance spectroscopy at cryogenic temperatures to monitor the reduction state of the cluster. Laser flash photolysis experiments have been used to monitor electron transfer from reduced ferredoxin to nitrite reductase and have provided the first evidence for electron transfer between the two prosthetic groups of the enzyme. The effect of ionic strength on the observed kinetics has provided support for the proposal that electrostatic interactions between ferredoxin and nitrite reductase play an important role in the reaction mechanism.

Kinetics↗
PubMed1994

Solid state investigation of mefloquine hydrochloride.

Mefloquine hydrochloride was crystallized under different conditions and the products were studied by thermal analysis, IR spectroscopy and X-ray diffraction. It was demonstrated that different conditions of crystallization resulted in different crystal structures. The alpha- and delta-modifications were hydrates, the beta- and gamma-forms were polymorphs, the epsilon-form was an acetone solvate and theta-mefloquine was tetrahydrofuran solvate. During long storage at room temperature, the alpha-, delta- and theta-forms of mefloquine hydrochloride were transformed. By heat treatment of the delta-, epsilon and theta-modifications, a new crystal structure was obtained.

Calorimetry, Differential Scanning↗
PubMed1993

Amino acid residues in Anabaena ferredoxin crucial to interaction with ferredoxin-NADP+ reductase: site-directed mutagenesis and laser flash photolysis.

Ferredoxin (Fd) functions in photosynthesis to transfer electrons from photosystem I to ferredoxin-NADP+ reductase (FNR). We have made several site-directed mutants of Anabaena 7120 Fd and have used laser flash photolysis to investigate the effects of these mutations on the kinetics of reduction of oxidized Fd by deazariboflavin semiquinone (dRfH.) and the reduction of oxidized Anabaena FNR by reduced Fd. None of the mutations influenced the second-order rate constant for dRfH. reduction by more than a factor of 2, suggesting that the ability of the [2Fe-2S] cluster to participate in electron transfer was not seriously affected. In contrast, a surface charge reversal mutation, E94K, resulted in a 20,000-fold decrease in the second-order rate constant for electron transfer from Fd to FNR, whereas a similar mutation at an adjacent site, E95K, produced little or no change in reaction rate constant compared to wild-type Fd. Such a dramatic difference between contiguous surface mutations suggests a very precise surface complementarity at the protein-protein interface. Mutations introduced at F65 (F65I and F65A) also decreased the rate constant for the Fd/FNR electron transfer reaction by more than 3 orders of magnitude. Spectroscopic and thermodynamic measurements with both the E94 and F65 mutants indicated that the kinetic differences cannot be ascribed to changes in gross conformation, redox potential, or FNR binding constant but rather reflect the protein-protein interactions that control electron transfer. Several mutations at other sites in the vicinity of E94 and F65 (R42, T48, D68, and D69) resulted in little or no perturbation of the Fd/FNR interaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1993

Direct measurement of cyclic current-voltage responses of integral membrane proteins at a self-assembled lipid-bilayer-modified electrode: cytochrome f and cytochrome c oxidase.

Direct cyclic voltage-current responses, produced in the absence of redox mediators, for two detergent-solubilized integral membrane proteins, spinach cytochrome f and beef heart cytochrome c oxidase, have been obtained at an optically transparent indium oxide electrode modified with a self-assembled lipid-bilayer membrane. The results indicate that both proteins interact with the lipid membrane so as to support quasi-reversible electron transfer redox reactions at the semiconductor electrode. The redox potentials that were obtained from analysis of the cyclic "voltammograms," 365 mV for cytochrome f and 250 and 380 mV for cytochrome c oxidase (vs. normal hydrogen electrode), compare quite well with the values reported by using conventional titration methods. The ability to obtain direct electrochemical measurements opens up another approach to the investigation of the properties of integral membrane redox proteins.

Conductometry↗
PubMed1993

Purification and properties of an unusual membrane-derived cytochrome b-561 from the purple phototrophic bacterium Rhodobacter capsulatus, which is structurally related to the bacteriochlorophyll-binding protein, LHII beta.

An abundant cytochrome b-561 was solubilized from Rhodobacter capsulatus membranes by successive treatments with perchlorate and butanol/water. Neither procedure was effective alone although they could be combined into a single step. Once solubilized, cytochrome b-561 was purified by standard chromatographic procedures used for water-soluble proteins without addition of butanol or detergents. Cytochrome b-561 appears to be highly acidic, it has a size greater than about 1000 kDa as isolated, and the subunit size measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is less than 8 kDa. The redox potential measured by cyclic voltammetry is -65 mV at pH 7. The N-terminal amino acid sequence is identical to that of the Rb. capsulatus LHII beta light-harvesting bacteriochlorophyll binding protein subunit which has only 48 amino acid residues, and the mass, determined by mass spectroscopy, is identical to that of LHII beta. There is but one heme per two to three peptide chains of 5 kDa, which suggests that the two extraplanar ligands to the heme are on separate subunits. There is strong exciton splitting in the circular dichroism spectrum in the Soret region indicative of heme-heme interaction. The helix content based on far-uv CD is 41%. Together, these properties of cytochrome b-561 are very similar to those of isolated LHII alpha beta bacteriochlorophyll-protein complexes.

Amino Acid Sequence↗
PubMed1992

Direct electrochemistry of thioredoxins and glutathione at a lipid bilayer-modified electrode.

By using direct electrochemical analysis we have established that the reduction of Escherichia coli thioredoxin (EcT), T4 thioredoxin (T4T), and glutathione (GSSG) occurs at a self-assembled lipid bilayer-modified gold electrode via two separate one-electron processes. The first electron transfer has half-wave potentials of -0.05 +/- 0.01, -0.07 +/- 0.01, and -0.06 +/- 0.01 V, whereas the second one has values of -0.48 +/- 0.01, -0.39 +/- 0.01, and -0.45 +/- 0.01 V, for EcT, T4T, and GSSG, respectively. The scan-rate dependence of the cyclic voltammetry indicates, for both waves, that the process of electron transfer is dominated by a bulk diffusion of free species to and from the electrode, and that strongly adsorbed species do not significantly contribute at the scan rates used. The voltage separation of the peak currents indicates a quasi-reversible electron transfer process with an electrochemical rate constant which is larger for the second (lower potential) electron than for the first one. Using the above half-wave potentials of the one-electron steps, one can calculate a thermodynamic half-wave potential for the two-electron reduction processes. The values of these potentials are -0.265, -0.23, and -0.25 V for EcT, T4T, and GSSG, respectively. These are in excellent agreement with literature values obtained from equilibrium measurements of enzyme-catalyzed reactions involving these species. It is quite clear from these results that lipid bilayer-modified electrodes provide a biocompatible and direct means of efficiently carrying out electrochemical reactions with sulfur-based redox systems, as we have previously shown to be the case with metalloproteins.

Electrochemistry↗
PubMed1992

Direct electrochemistry of spinach plastocyanin at a lipid bilayer-modified electrode: cyclic voltammetry as a probe of membrane-protein interactions.

The electron transfer reactions between a lipid bilayer-modified gold electrode and oxidized spinach plastocyanin have been studied by cyclic voltammetry, using either an electrically neutral phosphatidylcholine (PC) bilayer or a positively charged PC bilayer containing 40 mol% dimethyldioctadecylammonium chloride, at two ionic strengths of electrolyte (0.02 and 0.2 M NaClO4). Plastocyanin was found to interact strongly enough with the lipid membrane to support an efficient electron transfer reaction with the electrode. The interaction forces, and therefore the mode of diffusion of plastocyanin molecules to the electrode, which limits the electron transfer rate, could be controlled by the PC concentration. At low lipid concentrations (0-5 mg/ml), electrostatically attractive interactions between specific microelectroactive sites on the surface of the lipid membrane and plastocyanin molecules predominate, producing a radial mode of diffusion of the protein molecules to the electrode surface. On the other hand, at high lipid concentrations (greater than 5 mg/ml), interaction between plastocyanin and the lipid membrane occurs via hydrophobic forces, and a linear diffusion of protein molecules limits the electron transfer process. These observations support and extend other experimental and theoretical results which indicate two possible sites on the surface of the plastocyanin molecule, one hydrophobic and one negatively charged, which are able to participate in electron transfer reactions. We conclude that electrochemical measurements with the present system provide a new approach to the study of redox protein-membrane interactions.

Electrochemistry↗
PubMed1991

A self-assembled pigmented BLM on a platinum support: the light-induced electrical effects.

The light-induced voltage and current changes under continuous illumination have been investigated in pigmented self-assembled lipid bilayer membranes deposited on a platinum electrode. Such self-organized pigmented bilayer-platinum system containing Zn-Phthalocyanine (ZnPc) as a photosensitizer and glycerol-dioleate (GDO) as a bilayer forming solution has been found to shift its electrode potential to more positive value on light irradiation as well as to increase the cathodic current across the membrane. The results indicate a direct electron transfer from the platinum electrode to hydrogen ion in the electrolyte solution. Furthermore, it has also been demonstrated a dramatic increase of the photocurrent over the time course of BLM formation visualizing a role of the bulk quenching processes which are significantly diminished in thin bilayer membrane.

Diglycerides↗
PubMed1991

Lipid bilayer-based sensors and biomolecular electronics.

The lipid bilayer postulated as the basic structural matrix of biological membranes is widely accepted. Experiments in the early 1960s have made direct studies of lipid bilayers possible. At present, the planar bilayer lipid membrane (BLM) together with spherical lipid bilayers (liposomes), upon suitable modification, serves as a most appropriate model for biological membranes. In recent years, advances in microelectronics and interest in ultrathin organic films, including BLMs, have resulted in a unique fusion of ideas toward the development of biosensors and transducers. Furthermore, recent trends in interdisciplinary studies in chemistry, electronics, and biology have led to a new field of research: biomolecular electronics. This exciting new field of scientific-technological endeavor is part of a more general approach toward the development of a new, postsemiconductor electronic technology, namely, molecular electronics with a long-term goal of molecular computers. Experimental BLMs have been mainly used in the past as models of biological membranes. The methods of BLM studies may not be familiar to those outside biomedical research. Therefore, a brief description of the experimental techniques will be given in Section IV. Recently, it has been demonstrated that BLMs, after suitable modification, can function as electrodes and exhibit nonlinear electronic properties. These and other experimental findings relevant to sensor development and to "biomolecular electronic devices" (BED) will be covered in Section V, after a brief description of biomembranes which have been suggested as nature's molecular devices (Section III). In the last section, the potential use of the BLM system together with its modifications in the development of a new class of organic diodes, switches, biosensors, electrochemical photocells, and biofuel cells will be presented (Section VI). Additionally, this paper, besides presenting a review of our work and those of others on BLMs and liposomes in relation to biosensors and molecular electronics, reports a novel technique for obtaining BLMs (or lipid bilayers) on solid supports. The presence of solid support on one side of the BLM greatly enhances its mechanical stability, while retaining the dynamic properties of the lipid bilayer. Advantages of the new technique for self-assembling amphiphilic molecules on rigid substrates are discussed in terms of their possible uses. That is, the new BLM system (s-BLMs) is potentially useful for technological applications in the area of biosensors, enzyme electrodes, and molecular electronics as well as biochips (Section IV.C). The dividing line between the present microscopic and the future molecular electronics is 1 micron.(ABSTRACT TRUNCATED AT 400 WORDS)

Biological Transport, Active↗
PubMed1990

Self-assembling bilayer lipid membranes on solid support.

Solid-supported bilayer lipid membranes (s-BLMs) that possess some properties similar to those of conventional BLMs can be self-assembled on a freshly cleaved metal wire by a two-step procedure: (i) The tip of a Teflon-coated platinum wire, while immersed in a lipid solution, is cut off with a scalpel; (ii) the new tip of the wire, having become coated with lipid solution, is transferred into 0.1 M KCl. After a few minutes, a stable lipid bilayer forms spontaneously on the tip of the wire, as verified by electrical measurements. An application of such a supported BLM (s-BLM) is reported for the detection of Pb2+ ions. The s-BLM is liquid-crystalline in structure, which makes it amenable to modification for basic studies, as well as for technological applications such as biosensors and molecular electronic devices.

Lipid Bilayers↗
PubMed1989

Deposition of a photosensitive complex within a lecithin bilayer lipid membrane.

In this paper the phenomenon of a photosensitive ion complex of Brilliant Yellow and ferric ions formation in the electrolyte phase and its subsequent deposition within a bilayer lipid membrane (BLM) is described. Deposition of light sensitive complex into the BLM considerably increases its mechanical stability and drastically changes its electrochemical and photoelectrical properties as well.

Azo Compounds↗