PubMed Health⌕ Search

Biomedical subjects

F M Goñi

Publications and source records attributed to F M Goñi.

At least 73 records · Page 4Linked to original sources
PubMed1995

Topological properties of two cubic phases of a phospholipid:cholesterol:diacylglycerol aqueous system and their possible implications in the phospholipase C-induced liposome fusion.

Water dispersions of phospholipid:cholesterol:diacylglycerol may, under certain conditions, originate either the lipid- and water-permeable Q224 cubic phase, or the lipid-permeable but water-impermeable Q227 cubic phase. These results are discussed within the framework of the phospholipase C-induced fusion of liposomes [Nieva et al. (1993) Biochemistry 32, 1054]. It is suggested that the cubic phases Q224 and Q227 represent two classes of lipid organization, one promoting, the other hindering the mixing of aqueous contents that is characteristic of membrane fusion. In this context, inverted micelles appear to be the end point of the fusion process, rather than fusion intermediates.

Bacillus cereus↗
PubMed1995

A pathway for the thermal destabilization of bacteriorhodopsin.

A variety of structural techniques, including IR spectroscopy, reveals that thermal denaturation of bacteriorhodopsin follows a given pathway (successively rearrangement of helical structures, extensive deuterium exchange, and finally protein aggregation) irrespective of heating rate, pH or ionic strength conditions. In all cases, thermal denaturation leads to a 'compact denatured state' which retains a large proportion of ordered structure.

Bacteriorhodopsins↗
PubMed1995

An infrared investigation of palmitoyl-coenzyme A and palmitoylcarnitine interaction with perdeuterated-chain phospholipid bilayers.

Mixtures of di-(perdeuteropalmitoyl)-sn-glycero-3 choline ([2H62]Pam2GroPCho) with palmitoylcarnitine or palmitoyl-CoA in aqueous suspension have been examined by Fourier-transform infrared spectroscopy. The C-H (or C-D) region of the spectrum shows that an order-disorder transition exists in pure aqueous palmitoylcarnitine at 45 degrees C; palmitoylcarnitine mixes with [2H62]Pam2GroPCho without perturbing the gel-fluid transition of the phospholipid even at [2H62]Pam2GroPCho/palmitoylcarnitine 1:2 molar ratios; and palmitoyl-CoA, however, at similar proportions, smears out the [2H62]Pam2GroPCho transition as detected from C-D stretching vibrations. Relevant data from the carbonyl region include; the high-frequency (non-hydrogen bound) carbonyl subpopulation, but not the low frequency one, detects the gel-to-fluid transition of the phospholipid; the carbonyl region detects the thermotropic transition over a wider temperature range than the methylene stretching region, i.e. detects changes starting well below and ending several degrees above the methylene transition temperature, and a significant interaction may occur between some coenzyme A group and the carbonyl groups of the phospholipid. The latter interaction may contribute to explain the coenzyme A/carnitine exchange during mitochondrial fatty acid import.

Deuterium↗
PubMed1995

Electrokinetic charge of the anesthetic-induced bR480 and bR380 spectral forms of bacteriorhodopsin.

The translational and rotational electrokinetics of the anesthetic-induced spectral transitions bR568-->bR480-->bR380 of bacteriorhodopsin have been investigated. Formation of the bR480 form is associated with an increase of the purple membrane negative electrokinetic charge, while the transformation of bR480 into bR380 is accompanied by a decrease of the membrane negative charge as compared to that of the 480 nm-absorbing form. Removal of anesthetics leads to the back transitions bR480-->bR568 and (in part) bR380-->bR568; however, the electrokinetic charge of the native membranes is not restored. A strong decrease in the electric polarizability and the appearance of a slow polarizability component are also observed in anesthetic-treated membranes. Comparison with the electrokinetic behaviour of partially delipidated membranes and with that of liposomes composed of purple membrane total lipids suggests that: (i) anesthetic molecules partition mainly at the protein/lipid interface inducing irreversible rearrangement of the boundary lipid layer, and (ii) different mode(s) or site(s) of interaction are responsible for the spectral and surface charge effects. The data are compatible with the hypothesis of anesthetics acting through partial dehydration of the membrane surface.

Anesthetics↗
PubMed1995

Surface-core relationships in human low density lipoprotein as studied by infrared spectroscopy.

The secondary structure of human apolipoprotein B at 37 degrees C is estimated to be 24% alpha-helix, 23% beta-sheet, 6% beta-turns, 24% unordered structure, and 24% "beta-strands," characterized by a band around 1618 cm-1, and consistent with extended string-like chains in contact with the lipid moiety not forming beta-sheets. When cooled to a temperature below the cholesteryl ester transition at 30 degrees C, the ordering of the low density lipoprotein core results in reversible changes in the protein conformation, decreasing the apparent amount of alpha-helix, beta-strand, and unordered structure below 30 degrees C and increasing beta-sheet and beta-turns. Lowering the ionic strength affects the core-associated transitions, shifting their temperature from 30 to 20 degrees C, and modifying protein conformation below the transition. An additional thermal event is observed at 75 degrees C, leading to irreversible protein denaturation. In the broad temperature range between the 30 and 75 degrees C transitions, apolipoprotein B is stable toward both temperature and ionic strength changes. After thermal denaturation, the protein retains a certain degree of ordered structure.

Apolipoproteins B↗
PubMed1995

Liposome destabilization induced by the HIV-1 fusion peptide effect of a single amino acid substitution.

The 23-residue synthetic peptide representing the N-terminus of HIV-1 gp41 is known to induce either leakage or fusion of lipid vesicles depending on the experimental conditions. In this paper we report that a polar amino acid substitution V-->E at position 2, known to block gp41 activity in vivo, makes the peptide unable to destabilize and/or fuse membranes. Moreover this variant, unlike the parent peptide, is never found in the membrane-associated beta conformation.

Amino Acid Sequence↗
PubMed1995

The binding of divalent cations to Escherichia coli alpha-haemolysin.

alpha-haemolysin, an extracellular protein toxin of Escherichia coli, is known to disrupt eukaryotic cell membranes. In spite of genetic evidence of Ca(2+)-binding motifs in its sequence, conflicting results are found in the literature on the requirement of divalent cations for the membranolytic activity of the toxin. Moreover, Ca(2+)-binding sites have not been characterized to date in the native protein. The results in this paper show that when Ca2+ levels are kept sufficiently low during bacterial growth and toxin purification, membrane lysis does not occur in the absence of added divalent cations. Ca2+ and, at higher concentrations, Sr2+ and Ba2+, support the lytic activity, but Mg2+, Mn2+, Zn2+ and Cd2+ appear to be inactive in this respect. Binding of metal ions can be followed by changes in the intrinsic fluorescence of alpha-haemolysin; ions supporting lytic activity produce changes in the intrinsic fluorescence that are not caused by the inactive ones. Scatchard analysis of 45Ca2+ binding reveals three equivalent, independent sites, with Kd approximately 0.11 mM. No 45Ca2+ binding is observed when the protein is incubated with Zn2+; conversely, incubation with Ca2+ prevents subsequent binding of 65Zn2+. In the light of three-dimensional data available for a structurally related protein, alkaline protease of Pseudomonas aeruginosa [Baumann, U., Wu, S., Flaherty, K. M. & McKay, D. B. (1993) EMBO J. 12, 3357-3364] it is suggested that alpha-haemolysin may bind a larger number of Ca2+ than the three that are more easily exchangeable and are thus detected in the 45Ca(2+)-binding experiments. In addition, structural similarities and conservation of ion-binding motifs support the hypothesis that His 859 is involved in the mutually exclusive binding of Zn2+ and Ca2+.

Amino Acid Sequence↗
PubMed1995

Differential penetration of fatty acyl-coenzyme A and fatty acylcarnitines into phospholipid monolayers.

The ability of fatty acyl-CoA's and fatty acylcarnitines to penetrate phospholipid monolayers was comparatively studied, in view of the important role of both kinds of derivatives in fatty acid transport across mitochondrial membranes. The interaction occurs predominantly through hydrophobic forces. Acylcarnitines penetrate phospholipid monolayers more strongly than acyl-CoAs; in addition the former show a positive cooperativity when they bind to the interface. These properties would facilitate membrane transfer of fatty acylcarnitines over that of their CoA homologues.

Acetylcarnitine↗
PubMed1994

Early and delayed stages in the solubilization of purple membrane by a polyoxyethylenic surfactant.

The purpose of this paper is to explore the reasons by which purple membrane solubilization by detergents takes hours, or even days, to reach equilibrium, while most biomembranes are solubilized in a matter of seconds, or minutes. With that aim, changes in the purple membrane absorption spectrum produced by hydrogenated Triton X-100 under equilibrium conditions (24 h) have been compared to those caused by the same surfactant in the minute, second and sub-second time scale. It is found that the various processes that accompany, or lead to, solubilization are already detected, and even reach an apparent equilibrium, in the 10 s that follow detergent addition. No new phenomena are detected in the following minutes, or hours, that are relevant to the process under study. This leads to the conclusion that the long solubilization process consists of the repeated operation of simple phenomena that are relatively fast in themselves. A hypothesis is proposed according to which the tight crystalline organization of the purple membrane prevents the insertion of detergent monomers in the lipid bilayer; instead, the surfactant would bind the periphery of the patches, i.e., the hydrocarbon-water contact region, and solubilization would take place gradually, from the periphery towards the core of the membrane patches, at a progressively lower rate as the amounts of free detergent and detergent-binding sites are decreased by the previous solubilization steps.

Kinetics↗
PubMed1994

Structure and thermal denaturation of crystalline and noncrystalline cytochrome oxidase as studied by infrared spectroscopy.

Fourier-transform infrared spectroscopy has been applied to the study of lipid vesicle-supported two-dimensional crystals and noncrystalline preparations of beef heart cytochrome oxidase. At room temperature, no conformational differences are seen between the noncrystalline and crystalline proteins, whose conformation is shown to consist of ca. 40% alpha-helix, 20% extended structures (including beta-sheet), 17% beta-turns, and 22% open loops plus nonstructured conformations. A novel infrared approach that combines quantitative spectral band decomposition with the study of the thermal behavior of each component has been applied. The procedure allows the independent examination of temperature-induced changes in individual structural elements (alpha-helix, beta-sheet, beta-turns, and unordered). All these reflect, upon heating the protein from 20 to 80 degrees C, a major irreversible thermal event centred at 55-60 degrees C, leading to a molecular state devoid of enzyme activity but with a defined secondary structure; in addition, when the band position, percent area (integrated intensity), and bandwidth of the various amide I components are separately plotted versus temperature, each component is seen to behave in a characteristic way. Thermal denaturation in D2O buffer shows a decrease in nonstructured conformations and an increase in beta-turns without major changes in the proportion of alpha-helix. Temperature-induced changes are not the same in amorphous and crystalline structures, the latter being in general more stable toward the thermal challenge. The above data extend and confirm previous structural studies on cytochrome oxidase using cryo-electron microscopy.

Animals↗
PubMed1994

Thermodynamic and structural stability of cytochrome c oxidase from Paracoccus denitrificans.

The structural stability of the integral membrane protein cytochrome c oxidase from Paracoccus denitrificans has been measured by high-sensitivity differential scanning calorimetry and Fourier transform infrared spectroscopy. Contrary to the mammalian enzyme or the yeast enzyme, which are composed of 13 subunits, the bacterial enzyme has only three or four subunits, thus providing a unique opportunity to examine the magnitude of the forces that stabilize this enzyme and to establish accurate structural assignments of events observed calorimetrically. In this paper, experiments have been performed with the wild-type enzyme and with a mutant enzyme lacking subunit III. Our results show that subunits I and II form a highly cooperative complex which denatures as a single cooperative unit at 67 degrees C, while subunit III is less stable and denatures 20 degrees C earlier. Reduction of the enzyme causes a large increase in the stability of subunits I and II but has absolutely no effect on subunit III. Despite the lack of a strong interaction between subunit III and the catalytic subunits, the absence of subunit III leads to a turnover-induced loss of electron-transfer activity. The magnitude of the energetic parameters and the infrared spectroscopic experiments indicate that the enzyme does not completely unfold upon thermal denaturation and that significant degrees of structure are preserved. The amount of native alpha-helix structure, which is 45% in the native state, decreases only to 30% after thermal denaturation. Presumably, the residual helical structure existing after thermal denaturation belongs to the intramembranous portions of the protein. The calorimetric behavior of subunit III does not fully conform to that expected for a highly alpha-helical membrane protein. The picture that emerges from these experiments is that, in the temperature-denatured form of the enzyme, most of the extramembranous structural elements are denatured while most of the intramembranous secondary structure is maintained even though native tertiary interactions appear to be disrupted.

Calorimetry, Differential Scanning↗
PubMed1994

An assessment of the biochemical applications of the non-ionic surfactant Hecameg.

A number of properties and effects of the novel non-ionic detergent Hecameg (6-O-(N-heptylcarbamoyl)-methyl-alpha-D-glucopyranoside) have been examined in view of its possible biochemical applications. In particular, its critical micellar concentration has been measured, and its effects on pure lipid membranes, soluble and membrane-bound enzymes have been recorded. Hecameg has some advantageous and some less advantageous properties; its relatively high critical micellar concentration (16.5 mM), almost insensitive to pH or ionic strength changes, makes it suitable for reconstitution procedures in which detergent must be removed by dialysis. It is also an effective lipid-solubilizing agent, producing leakage of vesicle contents at detergent concentrations well below the solubilizing range. Among the drawbacks, the presence of an amide group in the molecule may interfere with the protein amide group in spectroscopic measurements. It also appears to be less gentle than other nonionic surfactants towards certain enzyme activities.

Adenosine Triphosphatases↗
PubMed1994

Interaction of the HIV-1 fusion peptide with phospholipid vesicles: different structural requirements for fusion and leakage.

This paper presents a study on the membrane fusion activity of a 23-residue synthetic peptide, representing the N-terminus of gp41 of the human immunodeficiency virus type I (HIV-1; LAV1a strain), in a model system involving large unilamellar vesicles (LUV) composed of the negatively charged 1-palmitoyl-2-oleoylphosphatidylglycerol (POPG). The peptide (HIVarg) induced fusion of POPG LUV as evidenced by (i) mixing of membrane lipids, (ii) mixing of aqueous vesicle contents, and (iii) an irreversible increase in vesicle size. Fusion could be induced only in the presence of millimolar concentrations of Ca2+ or Mg2+, needed for induction of vesicle aggregation; the divalent cations by themselves did not induce any fusion. The rate constant of the fusion reaction, as determined by simulation of the process according to a kinetic model, increased dramatically with the peptide-to-lipid molar ratio, indicating that the peptide was the mediator of the process. In the absence of divalent cations, the HIVarg peptide induced leakage of small molecules due to formation of pores in the membrane of single vesicles. Final extents and kinetics of this leakage process could be simulated adequately by model calculations for peptide-to-lipid ratios ranging from 1:25 to 1:750. Experiments, in which the order of peptide and Ca2+ addition to the vesicles was varied, indicated that the peptide is likely to adopt two different structures, one in the absence of Ca2+, primarily supporting leakage by formation of pores in separate vesicles, and one in the presence of Ca2+, primarily supporting fusion. Once a final structure had been established, it persisted even upon addition or removal of Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium↗
PubMed1994

Real-time measurements of chemically-induced membrane fusion in cell monolayers, using a resonance energy transfer method.

Fusion of mouse melanoma cells grown in monolayers has been directly monitored by fluorescence resonance energy transfer between fluorescein and rhodamine probes attached to octadecanoic acid. Various poly(ethylene glycol)s (PEG), either alone or in combination with amphipathic molecules, have been used as fusogens. Fusion starts at a maximum rate as soon as PEG is removed from the medium and reaches a plateau after 20-30 min. Both the initial rate and extent of fusion have been recorded for each experiment. The extent of fusion shows in general a positive correlation with the initial rate, although PEGs with different molar masses appear to induce fusion at different rates, but to a similar extent. A good correlation has been found between the extent of fusion, as measured by fluorescence, and the 'fusion index' computed from cell and nucleus counting; a calibration curve is provided for the interconversion of both parameters. Optimum fusion values are obtained with 50% (w/v) PEG 1500. The effect of pre-treatments with surfactants (Triton X-100, sodium dodecylsulphate) on PEG-induced fusion has also been tested. Sodium dodecylsulphate, but not Triton, enhances considerably both the rate and extent of cell fusion. The in situ generation of the amphipathic molecule diacylglycerol, through the catalytic activity of a phospholipase C, also enhances significantly the fusion parameters. These results are in good agreement with previous studies based on syncytia counting.

Animals↗
PubMed1993

Fluorescence quenching at interfaces and the permeation of acrylamide and iodide across phospholipid bilayers.

Studies of fluorescence quenching in membrane proteins are complicated by the fact that the barrier effect of the bilayer towards the quenchers is not known with precision. Our studies show that (a) both acrylamide and iodide can permeate the membrane at comparable rates, (b) when quenchers are added externally to a vesicle suspension, the apparent Stern-Volmer quenching constants for the same fluorophores are lower in the inner than in the outer aqueous compartments, and (c) at least some non-polar fluorophores embedded in the bilayer are quenched by iodide, but not by acrylamide.

Acrylamide↗
PubMed1993

Time-resolved and equilibrium measurements of the effects of poly(ethylene glycol) on small unilamellar phospholipid vesicles.

The effects of poly(ethylene glycol) (PEG) on sonicated unilamellar vesicles made of phosphatidylcholine have been examined. Stopped-flow and equilibrium data are presented for vesicle aggregation, vesicle leakage, lipid mixing, and aqueous contents mixing. Vesicle aggregation is detected as a monoexponential increase in light scattering, for PEG concentrations between 5 and 10%. In the region between 10-15% and 23-27% PEG, under our experimental conditions, the increase in light scattering follows a more complex biexponential kinetics, and, under these conditions, vesicle aggregation is accompanied by lipid mixing, a combination of events denoted as "close apposition". Above 23-27% PEG, the increase in light scattering is accompanied by fast lipid mixing, and also mixing of aqueous contents, all this being indicative of vesicle fusion; in addition, leakage occurs under these circumstances. Fusion takes place at high PEG concentrations, as indicated above, without any dilution step. From a methodological point of view, the Tb/DPA assay is shown to be more appropriate than the ANTS/DPX method for leakage and fusion studies in the presence of PEG.

Energy Transfer↗