Göran Wikander

Human prostasome membranes exhibit very high cholesterol/phospholipid ratios yielding high molecular ordering.

Lipid analysis and ESR studies were carried out on prostasomes isolated from human semen. Cholesterol plus phospholipids amounted to approximately 0.80 mumol per mg protein with a striking quantitative domination of cholesterol over the phospholipids, the molar ratios of cholesterol/sphingomyelin/glycerophospholipids being 4:1:1. Saturated and monounsaturated fatty acids were dominating both in the glycerophospholipids and in sphingomyelin. The order parameters, S, deduced from ESR spectra of spin-labelled fatty acids incorporated into prostasome membranes order parameters, S, deduced from ESR spectra of spin-labelled fatty acids incorporated into prostasome membranes were very high, viz. 0.75 for 5-doxylstearic acid and 0.30 for 16-doxylstearic acid at 25 degrees C. Slightly lower values were obtained for the spin-labelled fatty acids when they were incorporated into dispersions of extracted prostasome lipids or into synthetic lipid mixtures of similar composition. The highly ordered lipids in the prostasome membrane thus seemed to be minimally perturbed by proteins in the membrane and ESR spectra showed no signs of immobilized lipids.

Normal Binding and Reactivity of Copper in Mutant Superoxide Dismutase Isolated from Amyotrophic Lateral Sclerosis Patients

Abstract: In some families with amyotrophic lateral sclerosis (ALS), the disease is linked to mutations in the gene encoding CuZn‐superoxide dismutase. The mutant CuZn‐superoxide dismutases appear to cause motor neuron degeneration by a toxic property, suggested to be linked to an altered reactivity of the active‐site Cu ions. Asp90Ala mutant CuZn‐superoxide dismutase was isolated from six patients with ALS, allowing properties of the mutant enzyme synthesized and conditioned in patients with ALS to be examined. The molecular mass of the Asp90Ala mutant CuZn‐superoxide dismutase was 45 Da lower than that of the wild‐type enzyme, as expected from the amino acid exchange. The mobility after sodium dodecyl sulfate‐polyacrylamide gel electrophoresis was markedly increased, however, suggesting altered properties of the polypeptide. The mutant CuZn‐superoxide dismutase showed a minimal reduction in stability but did not differ significantly from the wild‐type enzyme in enzymic activity, in content and affinity for active‐site Cu ions and in the propensity to catalyze formation of hydroxyl radicals. Our findings suggest that the deleterious effect of mutant CuZn‐superoxide dismutases on motor neurons in ALS is not related to altered reactivity of active‐site Cu ions, resulting in increased oxidant stress. Attention should therefore also be directed at other mechanisms and properties of the mutant polypeptides and their degradation products.

Induction of secondary structure in the peptide hormone motilin by interaction with phospholipid vesicles

Motilin is an intestinal peptide hormone that binds to a membrane bound receptor located in the gut tissue. Circular dichroism (CD) was used to study the interaction between either porcine or rabbit motilin or a 1-16 fragment of porcine motilin, with model systems of lipid membranes: sodium dodecyl sulphate (SDS), 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). The CD measurements show significant induction of secondary structure in both motilins and the fragment when negatively charged vesicles (DOPG) or negatively charged micelles (SDS) were present. In contrast, neutral DOPC vesicles did not induce any change in the secondary structure compared to water, in which a random-like secondary structure dominates. The induced secondary structure in the presence of DOPG vesicles is very close to that induced by a mixed aqueous solution containing 30% hexafluoroisopropanol, in which previous NMR-studies have resulted in a three-dimensional solution structure of porcine motilin. In both porcine and rabbit motilin the alpha-helix content is about 50%. This is in agreement with the presence of an amphipathic helix in the C-terminal half of motilin interacting with phospholipid membranes. The interaction appears to be mainly electrostatic in nature, and does not induce any significant alterations in the vesicle, as monitored by EPR studies of spin labels located at the fifth carbon atom of the backbone in a stearic acid molecule. In the 1-16 fragment the alpha-helical content induced by DOPG and SDS is only about 20%.

A quantitative electron‐spin resonance line shape study of the local motion in the micellar and liquid crystalline lamellar phases of the oleoyllysolecithin water system

The experimental electron‐spin resonance (ESR) line shapes of the micellar (L1) and lamellar (Lα) phases of the binary system 1‐oleoyl‐sn‐glycero‐3‐phosphocholine (OlLPC)/water have been interpreted quantitatively using a two‐dynamic (TD) model. This dynamic model comprises the local dynamics of Freed’s model [J. Chem. Phys. 55, 5270 (1971); 58, 3185 (1973); 77, 3915 (1982)] and a second Brownian motion referring to the combined effect of the micellar reorientation and the translational motion of the amphiphile molecule along the micellar surface. With this model, we focus our attention on a comparison of the local dynamics between the L1 and Lα phases. It is found from the TD model that the local ordering (λ) and dynamics (τ⊥) of the L1 phase change compared with those of the Lα phase according to λ(L1)≳λ(Lα) and τ⊥(L1)<τ⊥(Lα). The observed lower local ordering in the lamellar phase compared with that in the micellar phase may be interpreted in terms of the different packing constraints of the OlLPC mole...

Phase equilibria and formation of vesicles of dioleoylphosphatidylcholine in glycerol / water mixtures

The lipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) forms a lamellar liquid crystalline phase (L alpha) in arbitrary mixtures of glycerol and water. The phase has been characterized by means of X-ray diffraction, 31P-NMR spectroscopy and differential scanning calorimetry (DSC). In the L alpha state, and for DOPC concentrations greater than 50% (w/w), the thickness of the lipid bilayer decreases, while the area of the polar head group increases with increasing glycerol concentration. The phase transition from gel to L alpha state occurs in the range of 240 to 260 K. Contrary to a previous (McDaniel, R.V., McIntosh, T.J. and Simon, S.A. (1983) Biochim. Biophys. Acta 731, 97) study of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) we find that in the gel state, the thickness of the DOPC lipid bilayer is greater than that in the L alpha state. This suggests that in the gel state, the lipid acyl chains of DOPC are in extended configuration. The lamellar phase reaches its maximum swelling at about 50% (w/w) of DOPC. At lower DOPC concentrations a two-phase system is formed where the lamellar phase exists in equilibrium with excess of solvent. Unilamellar vesicles can be prepared from a diluted suspension of the lamellar phase either by using the sonicator or extruder technique. We show this by means of 31P-NMR, EPR and fluorescence spectroscopy. The mean radius of the vesicles, prepared by a sonicator, has been determined at different glycerol/water mixtures. It is found to decrease continuously from 100 A at 100% water to a minimum of 75 A at about 50% water in the solvent mixture. By further decreasing the water content in the solution, the radius rapidly increases, and a mean radius of 450 A is estimated at a water content of 10%. The rotational relaxation times of a fluorescent probe and two EPR spin probes, solubilized in DOPC vesicles, have been measured at different glycerol/water mixtures. It is found that the rotational rates are always much slower in the systems containing glycerol.

The influence of chlorine substituted benzenes on the formation of positronium in solutions of cyclohexane and 2, 2, 4-trimethylpentane

Abstract Inhibition of positronium formation caused by different chlorine substituted benzenes used as additives in cyclohexane and 2, 2, 4-trimethylpentane has been investigated by means of positron lifetime measurements. Comparisons with earlier results obtained in benzene as solvent show that mono-, di- and some of the isomeric tri-substituted forms of the additives behave similarly in the three solvents, while the inhibitor efficiency of the higher chlorinated substrate molecules is considerably less in the solvents under investigation. The results are discussed in terms of the “hot Ps-model” and the “spur-model”.

Effect of glycerol on the translational and rotational motions in lipid bilayers studied by pulsed-field gradient 1H NMR, EPR and time-resolved fluorescence spectroscopy

Glycerol can replace water in both lipid vesicles and lyotropic liquid-crystalline phases. 1,2-Dioleoyl-sn-glycero(3)phosphocholine (DOPC) forms a lamellar (Lα) liquid-crystaline phase in arbitrary mixtures of glycerol and water (Biochim. Biophys. Acta, 1993, 1149, 285.). Monoolein (MO) forms Lα and also cubic liquid-crystalline phases in glycerol–water mixtures. The present study is focussed on characterizing the influence of glycerol on the molecular dynamics in the lipid bilayer. By EPR and time-resolved fluorescence spectroscopy we measure the rotational mobility of spin-labelled fatty acids [2-(3-carboxypropyl)-4, 4-dimethyl-2-tridecyloxazolidin-3-yloxyl (5-DS) and 2-(14-carboxytetradecyl)-2-ethyl-4, 4-dimethyloxazolidin-3-yloxyl (16-DS)] and a hydrophobic fluorophore, 2, 5, 8, 11-tetra-tert-butylperylene (TBPe), respectively. The translational diffusion of MO in the cubic phase is obtained by pulsed-field gradient 1H FT NMR experiments. The rotational rate of 16-DS and TBPe decreases continuously with increasing glycerol concentration, being a factor of 2–3 lower at 100 % glycerol. A continuous decrease in the lipid translational diffusion coefficient, D, is also found with increasing glycerol content, so that D= 12.6 × 10–12 m2 s–1 at 0 % and D= 1.9 × 10–12 m2 s–1 at 100 % glycerol. The effects of glycerol on both the translational diffusion of the lipid in the bilayers and the rotational dynamics of the probe molecules residing in the interior of the hydrophobic regions are ascribed to changes of the viscosity in the interbilayer regions.

Inhibition of Ps formation in benzene and cyclohexane by CH3Cl and CH3Br. Correlation to radiation chemistry results

Abstract Positron-annihilation lifetime spectra have been measured for mixtures of CH 3 Cl and CH 3 Br in cyclohexane and of CH 3 Cl in benzene. The ortho-positronium (Ps) yield decreased monotonically from 38% and 43% in cyclohexane and benzene respectively to 11% in pure CH 3 Cl and 6% in pure CH 3 Br. The strength of the inhibition of Ps formation by CH 3 Br was ten times that of CH 3 Cl in cyclohexane, because the CH 3 Br − anion debrominates rapidly, while CH 3 Cl − is long-lived (= 30 ns) compared to the maximum time of Ps formation of 400–500 ps. as shown in radiation chemistry. The positron can pick off the electron from the CH 3 X − anions to form Ps. while it forms a bound state with the halides. X − . CH 3 Cl was a roughly three times weaker Ps inhibitor in benzene than in cyclohexane, which shows that CH 3 Cl − does not dechlorinate in times comparable to or shorter than 400–500 ps in benzene. An improved model for the explanation of Ps formation in mixtures, where the Ps yield versus electron scavenger concentration has a minimum, is proposed and discussed.

Inhibition and enhancement of positronium formation in solutions of fluorobenzenes

Abstract The influence of fluorobenzenes on the formation of o -Ps in the nonpolar solvents benzene, 2,2,4-trimethylpentane and cyclohexane has been studied. In the fluorobenzene-cyclohexane system most of the additives give rise to a powerful enhancement of the o -Ps yield compared to pure solvent. This promoting behaviour of the substrates becomes much less pronounced in the other two solvents investigated. o -C 6 H 4 F 2 is the only additive which behaves as a typical inhibitor of o -Ps formation. The results are discussed in terms of existing models for formation of positronium.

Inhibition, antiinhibition and enhancement of positronium formation in some different hydrocarbon—solute mixtures

Abstract Positron annihilation measurements have been performed in non-polar solvents with additions of C 6 H 5 Cl, C 6 H 5 CH 2 Cl, C 6 F 12 /CCl 4 and three different amines, DMA, TPA and TMPD. The halogen derivatives of benzene behave like typical inhibitors. In the “two-solte” system it appears that C 6 F 12 , which earlier has been shown to be a weak inhibitor, acts as an anti-inhibitor, when mixed with CCl 4 in cyclohexane solutions. The presence of the amines results in a powerful enhancement of the o -Ps yield in C 6 H 12 while this effect is strongly diminished in benzene solutions. The results are discussed and interpreted in terms of the spur reaction model of Ps formation.