George P. Kreishman

Ethanol-induced changes in neuronal membrane order. An NMR study

The effects of ethanol-d6 on the lipid matrix of rat brain neuronal membranes were investigated by delayed Fourier transform 1H-NMR techniques. At 24 degrees C, neither 0.1 nor 0.2% (v/v) ethanol-d6 measurably affected the methylene resonance intensity. However, 0.4 and 1.0% ethanol-d6 increased resonance intensity, 35 and 51%, respectively. With increasing temperature, a decrease in resonance intensity for 0.1% ethanol-d6 was observed reaching a maximum of 20% at 42 degrees C. Furthermore, increasing temperature attenuated the increases in resonance intensity seen with 0.4 and 1.0% ethanol-d6. At 24 degrees C, no concentration of ethanol-d6 had a significant effect on the choline methyl resonance. However, with increasing temperature both 0.1 and 0.2% ethanol-d6 decreased this resonances intensity. The intensity of the terminal methyl resonance was increased in a dose related fashion by ethanol-d6, reaching a maximum of +41% at 1.0% (24 degrees C). Increasing temperature attenuated this effect, but no concentration of ethanol-d6 significantly decreased resonance intensity. The increases and decreases in resonance intensity induced by ethanol-d6 are interpreted in terms of a decrease and an increase in membrane order, respectively. It is proposed that ethanol-d6 exerts two effects on neuronal membranes, an ordering effect on the membrane surface and a disordering effect in the membrane interior. A higher enthalpy of ethanol binding to the surface as compared to the interior of the membrane leads to an attenuation of the ethanol disordering effect with increasing temperature.

Determination of ethanol partition coefficients to the interior and the surface of dipalmityl-phosphatidylcholine liposomes using deuterium nuclear magnetic resonance spectroscopy

The binding of ethanol-d6 to dipalmityl-phosphatidylcholine liposomes (DPPC) can be separated into two processes, namely, ethanol in the bilayer and on the surface of the bilayer. For the deuterons of the methylene group, the T2 of both bound states is shorter than the respective preexchange lifetime (tau beta) and therefore the amount of ethanol bound to both sites can be determined from the decrease in the methylene intensity resonance in the presence of DPPC. For the methyl resonance, however, only the T2 of deuterons on ethanol bound to the surface is less than its tau beta and the amount of surface bound ethanol-d6 can be determined. Subtraction yields the amount of ethanol bound within the bilayer. The partition coefficient for internally bound ethanol remains constant from 0 to 3.5 m ethanol. Surface binding is, however, highly cooperative.

212 - The Effect of Bulk Solvent Structure on the Temperature Dependence of the Reduction Potential of Cytochrome c☆

Abstract The reduction potential of horse heart cytochrome c in various sodium halide solutions in H 2 O and D 2 O has been measured over the temperature range of 25 to 50 °C. In aqueous chloride solutions the temperature dependence was biphasic with the intersection point at 42 °C. This biphasic behavior is interpreted in terms of chloride-induced bulk destructuring of water at 42 °C. All samples in D 2 O and samples in H 2 O not containing chloride ion gave a linear temperature dependence. The decrease in reduction potential with increasing temperature for solutions containing F−, Cl−, Br− and I − correlates with the extent of anion binding to the oxidized form of cytochrome c .

Preliminary spectrofluoroelectrochemical studies indicate a possible conformational change in horse heart cytochrome c upon reduction

Abstract An optically transparent thin-layer electrode can be used in conjunction with a spectrofluorometer to monitor the changes in the fluorescence properties of tryptophan-59 of horse heart cytochrome c as a function of applied solution potential. The fluorescence spectrum for the reduced state differs from that of the oxidized state. Several factors attenuated the fluorescence of the tryptophan, namely, (1) energy transfer of the fluorescence to the heme and (2) because of the high concentrations used in these studies, the fluorescence is decreased by quenching by other cytochrome cs in solution. Taking these two factors into account, the observed fluorescence intensities at a given wavelength can be explained if one assumes a movement of the tryptophan toward the heme of 0.7 ± 0.3 A upon reduction.

The temperature dependence of the redox potential of horse heart cytochrome c in sodium chloride solutions.

The E0′ values for the conversion of horse heart cytochrome c from the oxidized to the reduced form as a function of temperature have been measured in 0.10 M NaCl, 0.10 M sodium phosphate, pH 7.0 solutions in H2O and D2O. In H2O, the decrease in the E0′ value is linear with increasing temperature up to 42°C. Above this temperature, the decrease is again linear but with a much greater slope. In D2O solutions, however, this biphasic behavior was not observed but instead a single line was obtained over the temperature range studied (25°C to 50°C). These results are interpreted in terms of the ability of NaCl to cause a destructuring of the bulk H2O above 42°C but not in the more stable D2O (Kreishman, Foss, Inoue and Leifer (1976) Biochemistry, 15, 5431–5435). This decrease in water structure results in a shift in the equilibrium to the larger oxidized form as indicated by the decrease in E0′.

Introduction of new Alcohol Binding Sites at the Lipid/Water Interface by the Incorporation of Monosialogangliosides into DPPC Liposomes

Following Overton’s early observation of the direct relationship between the efficacy of an alcohol and oil/water partition coefficient of that alcohol1, most research concerning the mechanism of action of ethanol and similar anesthetics has focused on the interior of neuronal membranes and model membrane systems. The lipid perturbation hypothesis simply states that the effects of alcohols result from changes in the fluidity of the interior of the membrane. The observed changes in membrane order, however, are usually small or occur at nonphysiological concentrations of alcohol2.

Guanine-uracil base-pairing

Abstract The interaction of guanosine and 2′-deoxyuridine has been examined by high resolution pmr spectroscopy in DMSO-water mixtures. Evidence is presented for G-U base-pairing in solvent mixtures where the water content is sufficiently high. Downfield shifts were observed for the N(1)-H, NH2 protons of G and the N(3)-H proton of U, suggesting that the complex formation involves three hydrogen-bonds and that the G base is pairing in the lactim-amino tautomeric structure. No evidence for G-U base-pairing in the “wobble” as well as other pairing schemes was obtained.

Identification of C31 and C32 Sterols in Pneumocystis carinii hominis‐Infected Human Lungs

SUMMARY Two sterols in autopsied whole lung specimens obtained from Pneumocystis carinii pneumonia patients were detected by gas‐liquid chromatography and their structures were elucidated by mass spectrometry and nuclear magnetic resonance spectrometry. Both were in the lanosterol series; the C31 sterol, with a methyl group at C‐24, was identified as euphorbol, and the more abundant C32 sterol, with an ethyl group at C‐24, is given the trivial name pnemocysterol.

Cyanogen-induced γ-glutamyl to imidazole cross-link in carbonic anhydrase: A unique mode of inhibition

The irreversible inhibition of carbonic anhydrase by cyanogen occurs by a unique mechanism. Cyanogen is an affinity label: it behaves like a carbodiimide and produces an intra‐molecular cross‐link without being incorporated. The nucleophile‐labile cross‐link is formed between a γ‐COOH of a Glu and an imidazole of a His with a 1:1:1 stoichiometry with the enzyme. The deletion of ~ 1 Glu and ~ 1 His was noted by amino acid analysis of enzymatically hydrolyzed carbonic anhydrase. The modified Glu was converted to 2,4‐diaminobutanoic acid and quantitated by amino acid analysis. The presence and quantity of modified His was supported through high‐resolution proton NMR analysis.The irreversible inhibition of carbonic anhydrase by cyanogen occurs by a unique mechanism. Cyanogen is an affinity label: it behaves like a carbodiimide and produces an intra-molecular cross-link without being incorporated. The nucleophile-labile cross-link is formed between a gamma-COOH of a Glu and an imidazole of a His with a 1:1:1 stoichiometry with the enzyme. The deletion of approximately 1 Glu and approximately 1 His was noted by amino acid analysis of enzymatically hydrolyzed carbonic anhydrase. The modified Glu was converted to 2,4-diaminobutanoic acid and quantitated by amino acid analysis. The presence and quantity of modified His was supported through high-resolution proton NMR analysis.

Ethanol disordering of GM1-enriched Short-Sleep synaptosomal plasma membranes

The Long-Sleep (LS) and Short-Sleep (SS) mouse synaptosomal plasma membranes differ in ethanol sensitivity at superficial membrane regions, which corresponds with the behavioral response of the mice to ethanol hypnosis. The only significant difference between these synaptosomal plasma membranes is the synaptosomal monosialoganglioside (GM1) content, LS > SS. Here, GM1 was examined as a parameter for increasing membrane sensitivity to ethanol effects in the ethanol-resistant SS membranes. Synaptosomal plasma membranes from SS mice were allowed to incorporate exogenous GM1. Membrane order was then studied at the surface, intermediate, and interior regions of the membranes by delayed Fourier transform proton NMR in the presence and absence of perdeuterated ethanol. Differences in membrane order were observed in all three membrane regions with increasing perdeuterated ethanol concentrations depending on the synaptosomal GM1 content.