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Biomedical subjects

D Marsh

Publications and source records attributed to D Marsh.

At least 271 records · Page 15Linked to original sources
PubMed1982

Gel-to-inverted hexagonal (L beta-HII) phase transitions in phosphatidylethanolamines and fatty acid-phosphatidylcholine mixtures, demonstrated by 31P-NMR spectroscopy and x-ray diffraction.

The phase behaviour of distearoyl- and dihexadecylphosphatidylethanolamine at high salt concentration and of dipalmitoylphosphatidylcholine + 66 mol% palmitic acid at pH 4.0 have been studied by high-field 31P-NMR spectroscopy and X-ray diffraction. In saturated NaCl, dihexadecylphosphatidylethanolamine undergoes a reversible transition at 74 degrees C directly from the lamellar gel phase L beta to the inverted hexagonal HII phase. A similar transition is observed at 78.4 degrees C for distearoylphosphatidylethanolamine in saturated NaCl (cooling scan). The single, sharp calorimetric transition at 61 degrees C observed in dipalmitoylphosphatidylcholine + 66 mol% palmitic acid at pH 4.0 also corresponds to a transformation directly from the lamellar gel to the inverted hexagonal phase, without an intervening fluid lamellar phase.

Calorimetry, Differential Scanning↗
PubMed1982

Spin label studies on osmotically-induced changes in the aqueous cytoplasm of Phaeodactylum tricornutum.

The effects of hyperosmotic stress and adaption on the aqueous cytoplasm of Phaeodactylum tricornutum have been studied with spin labels using 0.2M external Ni2+ to obtain spectra solely from labels within the cells. From partitioning of the TEMPO spin label between the internal aqueous phase and the membrane it is found that the internal volume of the cells decreased by approx. 50-60% in media of high osmotic strength (1.9 osmol/l). During the accumulation of proline in the cells (8.8 mg/ml packed cells) on incubation in the medium of high osmolarity for 3 days, the recovery of the volume was 80%. Further addition of proline to the medium resulted in an increase in the proline concentration in the cells (12.2 mg/ml packed cells) and a recovery in volume of 90%. Cells incubated in the absence of any nitrogen source showed very little recovery and were in a stressed state even in the absence of an osmotic gradient. From the rotational correlation times of the TEMPONE spin label it was found that the effective microviscosity in the cytoplasm of normal cells (approx. 3-8 cP) was considerably higher than that of the external medium (1 cP) and increased 1.5-2-fold under high osmotic stress (1.9 osmol/l). Adaption during the accumulation of proline only decreased the effective microviscosity by approx. 50% of the stressed-induced increase, a considerably smaller recovery than that of the cell volume.

Acclimatization↗
PubMed1982

ESR spin-label studies of lipid-protein interactions in membranes.

Lipid spin labels have been used to study lipid-protein interactions in bovine and frog rod outer segment disc membranes, in (Na+, K+)-ATPase membranes from shark rectal gland, and in yeast cytochrome oxidase-dimyristoyl phosphatidylcholine complexes. These systems all display a two component ESR spectrum from 14-doxyl lipid spin-labels. One component corresponds to the normal fluid bilayer lipids. The second component has a greater degree of motional restriction and arises from lipids interacting with the protein. For the phosphatidylcholine spin label there are effectively 55 +/- 5 lipids/200,000-dalton cytochrome oxidase, 58 +/- 4 mol lipid/265,000 dalton (Na+, K+)-ATPase, and 24 +/- 3 and 22 +/- 2 mol lipid/37,000 dalton rhodopsin for the bovine and frog preparations, respectively. These values correlate roughly with the intramembrane protein perimeter and scale with the square root of the molecular weight of the protein. For cytochrome oxidase the motionally restricted component bears a fixed stoichiometry to the protein at high lipid:protein ratios, and is reduced at low lipid:protein ratios to an extent which can be quantitatively accounted for by random protein-protein contacts. Experiments with spin labels of different headgroups indicate a marked selectivity of cytochrome oxidase and the (Na+, K+)-ATPase for stearic acid and for cardiolipin, relative to phosphatidylcholine. The motionally restricted component from the cardiolipin spin label is 80% greater than from the phosphatidylcholine spin label for cytochrome oxidase (at lipid:protein = 90.1), and 160% greater for the (Na+, K+)-ATPase. The corresponding increases for the stearic acid label are 20% for cytochrome oxidase and 40% for (Na+, K+)-ATPase. The effective association constant for cardiolipin is approximately 4.5 times greater than for phosphatidylcholine, and that for stearic acid is 1.5 times greater, in both systems. Almost no specificity is found in the interaction of spin-labeled lipids (including cardiolipin) with rhodopsin in the rod outer segment disc membrane. The linewidths of the fluid spin-label component in bovine rod outer segment membranes are consistently higher than those in bilayers of the extracted membrane lipids and provide valuable information on the rate of exchange between the two lipid components, which is suggested to be in the range of 10(6)-10(7) s-1.

Animals↗
PubMed1982

Melittin stimulates incorporation and degradation of sphingomyelin in synaptosomal plasma membranes.

Melittin enhanced sphingomyelin (SPM) degradation by the neutral membrane-bound sphingomyelinase from calf brain synaptosomal plasma membranes (SYM) up to 20-fold. Melittin in concentrations as high as 100 microM did not significantly alter membrane fluidity of SYM as measured by fluorescence depolarization and electron spin resonance (ESR) using diphenylhexatriene and a doxyl derivative of SPM, respectively. In the concentration range 100--1000 microM, melittin was observed to rigidify SYM. The incorporation of SPM derivatives into the lipid bilayer of SYM was demonstrated by ESR measurements. Melittin enhanced the uptake of SMP-derivatives into SYM.

Animals↗
PubMed1981

Titration of the phase transition of phosphatidylserine bilayer membranes. Effects of pH, surface electrostatics, ion binding, and head-group hydration.

The dependence of the gel-to-fluid phase transition temperature of dimyristoyl- and dipalmitoylphosphatidylserine bilayers on pH, NaCl concentration, and degree of hydration has been studied with differential scanning calorimetry and with spin-labels. On protonation of the carboxyl group (pK2app = 5.5), the transition temperature increases from 36 to 44 degrees C in the fully hydrated state of dimyristoylphosphatidylserine (from 54 to 62 degrees C for dipalmitoylphosphatidylserine), at ionic strength J = 0.1. In addition, at least two less hydrated states, differing progressively by 1 H2O/PS, are observed at low pH with transition temperatures of 48 and 52 degrees C for dimyristoyl- and 65 and 68.5 degrees C for dipalmitoylphosphatidylserine. On deprotonation of the amino group (pK3app = 11.55) the transition temperature decreases to approximately 15 degrees C for dimyristoyl- and 32 degrees C for dipalmitoylphosphatidylserine, and a pretransition is observed at approximately 6 degrees C (dimyristoylphosphatidylserine) and 21.5 degrees C (dipalmitoylphosphatidylserine), at J = 0.1. No titration of the transition is observed for the fully hydrated phosphate group down to pH less than or equal to 0.5, but it affinity for water binding decreases steeply at pH greater than or equal to 2.6. Increasing the NaCl concentration from 0.1 to 2.0 M increases the transition temperature of dimyristoyphosphatidylserine by approximately 8 degrees C at pH 7, by approximately 5 degrees at pH 13, and by approximately 0 degrees C at pH 1. These increases are attributed to the screening of the electrostatic titration-induced shifts in transition temperature. On a further increase of the NaCl concentration to 5.5 M, the transition temperature increases by an additional 9 degree C at pH 7, 13 degree C at pH 13, approximately 7 degree C in the fully hydrated state at pH 1, and approximately 4 and approximately 0 degree C in the two less hydrated states. These shifts are attributed to displacement of water of hydration by ion binding. From the salt dependence it is deduced that the transition temperature shift at the carboxyl titration can be accounted for completely by the surface charge and change in hydration of approximately 1 H2O/lipid, whereas that of the amino group titration arises mostly from other sources, probably hydrogen bonding. The shifts in pK (delta pK2 = 2.85, delta pK3 = 1.56) are consistent with a reduced polarity in the head-group region, corresponding to an effective dielectric constant epsilon approximately or equal to 30, together with surface potentials of psi congruent to -100 and -150 mV at the carboxyl and amino group pKs, respectively. The transition temperature of dimyristoylphosphatidylserine-water mixtures decreases by approximately 4 degree C each water/lipid molecule added, reaching a limiting value at a water content of approximately 9-10 H2O/lipid molecule.

Calorimetry, Differential Scanning↗
PubMed1981

Charge-induced tilt in ordered-phase phosphatidylglycerol bilayers evidence from X-ray diffraction.

X-ray diffraction studies have been performed, as a function of water content, on dipalmitoyl phosphatidylglycerol bilayers, both in the charged state at pH 8.0 and in the protonated state at pH 1.5, using buffers of 1.5 M salt concentration. Measurements were made at 20 degrees C, and the high-angle reflections indicated that the bilayers were in the ordered phase at both pH values. Lamellar diffractions were observed under all conditions studied. THe lamellar repeat reached a limiting value of 62.4 A (6.24 nm) at a water/lipid ratio of 0.24 at pH 8.0, and a limiting value of 67.3 A (6.73 nm) at a water/lipid ratio of 0.22 at pH 1.5. The area per lipid molecule in the plane of the bilayer, deduced from the bilayer thickness and the lipid partial specific volume, is 48 A2 (0.48 nm2) at pH 8.0 and 37 A2 (0.37 nm2) at pH 1.5. The area per molecule in the plane perpendicular to the chain axes, deduced from the X-ray short spacings, is 40.5 A2 (0.405 nm2) at pH 8.0 and 39.2 A2 (0.392 nm2) at pH 1.5. Thus the lipid molecules are tilted by approx. 30 degrees relative to the bilayer normal at pH 8.0, but are essentially untilted at pH 1.5.

Hydrogen-Ion Concentration↗
PubMed1981

Phospholipid chain immobilization and steroid rotational immobilization in acetylcholine receptor-rich membranes from Torpedo marmorata.

1. The ESR spectra of both phosphatidylcholine and phosphatidylethanolamine spin labels reveal an immobilized lipid component (tau R greater than or equal to 50 ns), in addition to a fluid component (tau R approximately 1 ns), in acetylcholine receptor-rich membranes prepared from Torpedo marmorata electroplax according to the method of Cohen et al. (Cohen, J.B., Weber, M., Huchet, M. and Changeux, J.-P. (1972) FEBS Lett. 26, 43--27). 2. The ESR spectra of the androstanol spin label display a component corresponding to molecules which are immobilized with respect to rotation about the long molecular axis (tau R greater than or equal to 50 ns), in addition to the fluid lipid bilayer component in which the molecules are rotating rapidly about their long axes (tau R approximately 1 ns). This immobilized component is observed throughout the temperature range 2--22 degrees C, at an approximately constant relative intensity of approx. 45% of the total, which is quantitatively the same as previously observed with fatty acid spin labels.

Androstanols↗
PubMed1981

Distinct states of lipid mobility in bovine rod outer segment membranes. Resolution of spin label results.

Freely diffusable lipid spin labels in bovine rod outer segment disc membranes display an apparent two-component ESR spectrum. One component is markedly more immobilized than that found in fluid lipid bilayers, and is attributed to lipid interacting directly with rhodopsin. For the 14-doxyl stearic acid spin label this more immobilized component has an outer splitting of 59 G at 0 degrees C, with a considerable temperature dependence, the effective outer splitting decreasing to 54 G at 24 degrees C. Spin label lipid chains covalently attached to rhodopsin can also display a two-component spectrum in rod outer segment membranes. In unbleached, non-delipidated membranes the 16-doxyl stearoyl maleimide label shows an immobilized component which has an outer splitting of 59 G at 0 degrees C and a considerable temperature dependence. This component which is not resolved at high temperatures (24--35 degrees C), is attributed to the lipid chains interacting directly with the monomeric protein, as with the diffusable labels. In contrast, in rod outer segment membranes which have been either delipidated or extensively bleached, a strongly immobilized component is observed with the 16-doxyl maleimide label at all temperatures. This immobilized component has an outer splitting of 62--64 G at 0 degrees C, with very little temperature dependence (61--62 G at 35 degrees C), and is attributed to protein aggregation.

Animals↗
PubMed1981

Saturation transfer ESR studies of molecular motion in phosphatidylglycerol bilayers in the gel phase: effects of pretransitions and pH titration.

The molecular motions of a phosphatidylglycerol spin label have been studied in dimyristoyl and dipalmitoyl phosphatidylglycerol bilayers below their ordered-fluid phase transition, both in the charged state at pH 8.0 and in the protonated state at pH 1.5. The saturation transfer ESR spectra, which are sensitive to motions in the correlation time range 10(-7)--10(-3) s, show clear distinctions in the molecular motion both between the charged and the protonated states and between the states above and below the pretransition in the charged bilayers. At low temperatures, below the pH 8.0 pretransition, the saturation transfer ESR spectra indicate rather similar motion at pH 8.0 and 1.5, with the rates being approx. 2-times faster at pH 8.0. The effective rotational correlation times are approx. 0.5--1.10(-4) and approx. 1--2.10(-4) s, deduced from the outer lineheight ratios which are sensitive only to motion of the long molecular axis, and approx. 0.2--1.10(-4) and 0.2--0.5.10(-4) s, deduced from the central lineheight ratio which is sensitive also to rotation around the long molecular axis, where the first and second values refer to dimyristoyl and dipalmitoyl phosphatidylglycerol, respectively. As the temperature is increased the outer lineheight ratios at both pH values remain constant until the main transition, indicating little or no increase in motion of the long axis. In contrast, the central lineheight ratio at pH 8.0 shows a sharp decrease before the main transition, corresponding to cooperative onset of rapid rotation around the long molecular axis, at or immediately below the pretransition. The effective correlation time of this long axis rotation is approx. 10(-7) s, two orders of magnitude faster than that below the pretransition. The bilayers at pH 1.5, for which no pretransition is detected, show only a slow, non-cooperative decrease in the central lineheight ratio with increasing temperature, rapid long axis rotation in dimyristoyl phosphatidylglycerol occurring only at the main transition. The onset of long axis rotation at pH 8.0 is also detected in the conventional spectra of a steroid spin label, which begins to rotate around its long molecular axis with a correlation time of approx. 10(-9) s whilst the bilayers are still in the gel phase. These observations further strengthen the homology between phosphatidylglycerols in the charged state and the corresponding phosphatidylcholines, and provide the dynamic counterpart of the pH-induced structural change previously observed in phosphatidylglycerol bilayers in the gel phase (Watts, A., Harlos, K., Maschke, W. and Marsh, D. (1978) Biochim. Biophys. Acta 510, 63--74).

Electron Spin Resonance Spectroscopy↗
PubMed1980

Lipid-lipid and lipid-protein interactions in chromaffin granule membranes. A spin label ESR study.

The ESR spectra of six different positional isomers of a stearic acid and three of a phosphatidylcholine spin label have been studied as a function of temperature in chromaffin granule membranes from the bovine adrenal medulla, and in bilayers formed by aqueous dispersion of the extracted membrane lipids. Only minor differences were found between the spectra of the membranes and the extracted lipid, indicating that the major portion of the membrane lipid is organized in a bilayer arrangement which is relatively unperturbed by the presence of the membrane protein. The order parameter profile of the spin label lipid chain motion is less steep over the first half of the chain than over the section toward the terminal methyl end of the chain. This 'stiffening' effect is attributed to the high proportion of cholesterol in the membrane and becomes less marked as the temperature is raised. The isotropic hyperfine splitting factors of the various positional isomers display a profile of decreasing polarity as one penetrates further into the interior of the membrane. No marked differences are observed between the effective polarities in the intact membranes and in bilayers of the extracted membrane lipids. The previously observed temperature-induced structural change occurring in the membranes at approx. 35 degrees C was found also in the extracted lipid bilayers, showing this to be a result of lipid-lipid interactions and not lipid-protein interactions in the membrane. A steroid spin label indicated a second temperature-dependent structural change occurring in the lipid bilayers at lower temperatures. This correspond to the onset of a more rapid rotation about the long axis of the lipid molecules at a temperature of approx. 10 degrees C. The lipid bilayer regions probed by the spin labels used in this study may be involved in the fusion of the chromaffin granule membrane leading to hormone release by exocytosis.

Adrenal Medulla↗