NMR studies of electrostatic potential distribution around biologically important molecules.
(17/1378)
A new experimental approach has been developed to study the distribution of local electrostatic potential around specific protons in biologically important molecules. The approach is the development of a method denoted as "spin label/spin probe," which was proposed by one of us (. Mol. Biol. 6:498-507). The proposed method is based upon the quantitative measurement of the contribution of differently charged nitroxide probes to the spin lattice relaxation rate (1/T1) of protons in the molecule of interest, followed by calculation of local electrostatic potential using the classical Debye equation. In parallel, the theoretical calculation of potential distribution with the use of the MacSpartan Plus 1.0 program has been performed. Application of the method to solutions of simple organic molecules (aliphatic and aromatic alcohols, aliphatic carboxylates (propionate anion), and protonated ethyl amine and imidazole) allowed us to estimate the effective potential around the molecules under investigation. These were found to be in good agreement with theoretically expected values. This technique was then applied to zwitterionic amino acids bearing neutral and charged side chains (glycine, lysine, histidine, and aspartic acid). The reliability of the general approach is proved by the data presented in this paper. Application of this new methodology can afford insight into the biochemical significance of electrostatic effects in biological systems. (+info)
Olfactory discrimination ability for homologous series of aliphatic alcohols and aldehydes.
(18/1378)
We tested the ability of human subjects to distinguish between members of homologous series of aliphatic alcohols (ethanol to n-octanol) and aldehydes (n-butanal to n-decanal). In a forced-choice triangular test procedure 20 subjects per series were repeatedly presented with all 21 binary combinations of the seven stimuli and asked to identify the bottle containing the odd stimulus. We found (i) that as a group, the subjects performed significantly above chance level in all tasks but two with the alcohols and all tasks but four with the aldehydes, and thus were clearly able to discriminate between most of the odor pairs presented; (ii) marked interindividual differences in discrimination performance, ranging from subjects who were able to significantly distinguish between all 21 odor pairs of a series to subjects who failed to do so with the majority of tasks; and (iii) a significant negative correlation between discrimination performance and structural similarity of odorants in terms of differences in carbon chain length for both homologous series. This suggests that carbon chain length may be one of presumably several determinants of the interaction between stimulus molecule and receptor, and thus may be a molecular property affecting odor quality of aliphatic alcohols and aldehydes. (+info)
Transport of water and glycerol in aquaporin 3 is gated by H(+).
(19/1378)
Aquaporins (AQPs) were expressed in Xenopus laevis oocytes in order to study the effects of external pH and solute structure on permeabilities. For AQP3 the osmotic water permeability, L(p), was abolished at acid pH values with a pK of 6.4 and a Hill coefficient of 3. The L(p) values of AQP0, AQP1, AQP2, AQP4, and AQP5 were independent of pH. For AQP3 the glycerol permeability P(Gl), obtained from [(14)C]glycerol uptake, was abolished at acid pH values with a pK of 6.1 and a Hill coefficient of 6. Consequently, AQP3 acts as a glycerol and water channel at physiological pH, but predominantly as a glycerol channel at pH values around 6.1. The pH effects were reversible. The interactions between fluxes of water and straight chain polyols were inferred from reflection coefficients (sigma). For AQP3, water and glycerol interacted by competing for titratable site(s): sigma(Gl) was 0.15 at neutral pH but doubled at pH 6.4. The sigma values were smaller for polyols in which the -OH groups were free to form hydrogen bonds. The activation energy for the transport processes was around 5 kcal mol(-1). We suggest that water and polyols permeate AQP3 by forming successive hydrogen bonds with titratable sites. (+info)
Effects of alcohol on the human placental GnRH receptor system.
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Isolation of human term placental membranes in the presence or absence of protease inhibitors indicated that protease inhibitors significantly reduced the amounts of [(125)I]-labelled gonadotrophin-releasing hormone (GnRH) binding to membrane GnRH-receptors in vitro by approximately 20%. This decrease was largely due to the ethanol used to dissolve the serine protease inhibitor, phenylmethylsulphonylfluoride (PMSF). Ethanol alone decreased the specific binding of [(125)I]-labelled GnRH isoform (IC(50), 7.9 +/- 0.8 mg/ml; n = 6) or agonist tracers (IC(50), 10.0 +/- 1.4 mg/ml; n = 6) to human placental membranes in a dose-dependent manner. Other alcohols also interfered with [(125)I]-GnRH isoform or agonist binding: inhibition increased with increasing carbon chain length and was dependent on the isomeric position of the hydroxyl group. Fractionation of term placental cytosol by gel chromatography demonstrated the presence of a high molecular weight fraction ( approximately 60-70 kDa) which inhibited [(125)I]-GnRH binding to human placental membranes. However, placental cytosol fractions did not cross-react significantly with a specific anti-GnRH antibody. Surprisingly, re-assay of cytosol fractions in the presence of a cocktail of protease inhibitors generated a factor (molecular weight approximately 40-50 kDa) which did cross-react strongly with the GnRH antibody. The generation of this factor was due to the ethanol solvent rather than to the protease inhibitors per se, as treatment of pooled 'latent' cytosol fractions with ethanol alone generated GnRH-like immunoactivity (irGnRH) which competed in parallel with GnRH standard. The amount of irGnRH generated depended on the concentration of ethanol added to the 'latent' cytosol fractions. However, ethanol had no effect on the assay in the absence of cytosol fraction, or with inactive cytosol fractions. Thus, ethanol can perturb the human placental GnRH/GnRH-receptor system in vitro in two distinct ways: by inhibition of GnRH binding to receptor, and by dissociation of complexed endogenous GnRH-like factor(s) from a GnRH-binding protein. It is postulated that high alcohol consumption in vivo may interfere with placental GnRH secretion/action and affect placental secretion of factors important to the establishment and maintenance of pregnancy. (+info)
The alkyl chain dependence of the effect of normal alcohols on agonist-induced nicotinic acetylcholine receptor desensitization kinetics.
(21/1378)
BACKGROUND: The nAcChoR is the prototypical member of a superfamily of ligand-gated ion channels that are all relevant targets of anesthetics and undergo desensitization upon prolonged exposure to agonist. This study was designed to investigate the effects of representative normal alcohols on the apparent rate of acetylcholine-induced nAcChoR desensitization. METHODS: Nicotinic acetylcholine receptors were obtained from the electroplax organ of Torpedo nobiliana. The apparent rate of acetylcholine-induced desensitization in the presence and absence of normal alcohols was measured using stopped-flow fluorescence. RESULTS: Normal alcohols as long as octanol (the longest studied) increased the apparent rate of desensitization induced by low concentrations of acetylcholine, shifting the agonist concentration-response curve for desensitization to the left Ethanol butanol, and, to a lesser extent, hexanol increased the maximal rate of desensitization induced by high, saturating concentrations of agonist. Beyond hexanol, heptanol and octanol had no effect on this maximal apparent rate of desensitization, even at concentrations that approach those that directly induce desensitization in the absence of agonist. CONCLUSION: Normal alcohols ranging from ethanol to octanol increase the apparent affinity of nAcChoR for agonist with potencies that are proportional to their hydrophobicities. However, normal alcohol effects on the rate constant for desensitization show a cutoff beyond hexanoL This suggests that the effects of normal alcohols on the apparent agonist affinity and rate constant for desensitization of nAcChoR may be modulated by distinct sites that have different steric constraints; the site(s) responsible for increasing the maximal rate of desensitization are predicted to be smaller than those that increase the apparent agonist affinity. (+info)
Ethanol and acetaldehyde elevate intracellular [Ca2+] and stimulate microneme discharge in Toxoplasma gondii.
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One of the first steps in host-cell invasion by the protozoan parasite Toxoplasma gondii occurs when the parasite attaches by its apical end to the target host cell. The contents of apical secretory organelles called micronemes have recently been implicated in parasite apical attachment to host cells. Micronemes are regulated secretory vesicles that discharge in response to elevated parasite intracellular Ca(2+) levels ([Ca2+]i). In the present study we found that ethanol and related compounds produced a dose-dependent stimulation of microneme secretion. In addition, using fluorescence spectroscopy on tachyzoites loaded with the Ca(2+)-sensitive fluorescent dye fura-2, we demonstrated that ethanol stimulated microneme secretion by elevating parasite [Ca2+](i). Furthermore, sequential addition experiments with ethanol and other Ca(2+)-mobilizing drugs showed that ethanol probably elevated parasite [Ca2+](i) by mobilizing Ca(2+) from a thapsigargin-insensitive compartment of neutral pH. Earlier studies have shown that ethanol also elevates [Ca2+](i) in mammalian cells. Thus, because it is genetically tractable, T. gondii might be a convenient model organism for studying the Ca(2+)-elevating effects of alcohol in higher eukaryotes. (+info)
Short-chain alcohols promote an early stage of membrane hemifusion.
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Hemifusion, the linkage of contacting lipid monolayers of two membranes before the opening of a fusion pore, is hypothesized to proceed through the formation of a stalk intermediate, a local and strongly bent connection between membranes. When the monolayers' propensity to bend does not support the stalk (e.g., as it is when lysophosphatidylcholine is added), hemifusion is inhibited. In contrast, short-chain alcohols, reported to affect monolayer bending in a manner similar to that of lysophosphatidylcholine, were here found to promote hemifusion between fluorescently labeled liposomes and planar lipid bilayers. Single hemifusion events were detected by fluorescence microscopy. Methanol or ethanol (1.2-1.6 w/w %) added to the same compartment of the planar bilayer chamber as liposomes caused a 5-50 times increase in the number of hemifusion events. Alcohol-induced hemifusion was inhibited by lysophosphatidylcholine. Promotion of membrane hemifusion by short-chain alcohol was also observed for cell-cell fusion mediated by influenza virus hemagglutinin (HA). Alcohol promoted a fusion stage subsequent to the low pH-dependent activation of HA. We propose that binding of short-chain alcohol to the surface of membranes promotes hemifusion by facilitating the transient breakage of the continuity of each of the contacting monolayers, which is required for their subsequent merger in the stalk intermediate. (+info)
Thermodynamics of alcohol-lipid bilayer interactions: application of a binding model.
(24/1378)
Several recent reports have provided evidence that interactions of small alcohols with lipid bilayer membranes are dominated by adsorption to the membrane-water interface. This mode of interaction is better modeled by binding models than solution theories. In the present study, alcohol-membrane interactions are examined by applying the 'solvent exchange model' [J.A. Schellmann, Biophys. Chem. 37 (1990) 121] to calorimetric measurements. Binding constants (in mole fraction units) for small alcohols to unilamellar liposomes of dimyristoyl phosphatidylcholine were found to be close to unity, and in contrast to partitioning coefficients they decrease through the sequence ethanol, 1-propanol, 1-butanol. Thus, the direct (intrinsic) affinity of the bilayer for these alcohols is lower the longer the acyl chain. A distinction between binding and partitioning is discussed, and it is demonstrated that a high concentration of solute in the bilayer (large partitioning coefficients) can be obtained even in cases of weak binding. Other results from the model suggest that the number of binding sites on the lipid bilayer interface is 1-3 times the number of lipid molecules and that the binding is endothermic with an enthalpy change of 10-15 kJ/mol. Close to the main phase transition of the lipid bilayer the results suggest the presence of two distinct classes of binding sites: 'normal' sites similar to those observed at higher temperatures, and a lower number of high-affinity sites with binding constants larger by one or two orders of magnitude. The occurrence of high-affinity sites is discussed with respect to fluctuating gel and fluid domains in bilayer membranes close to the main phase transition. (+info)