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D Marsh

Publications and source records attributed to D Marsh.

At least 217 records · Page 12Linked to original sources
PubMed1989

Spin-label ESR studies of lipid-protein interactions in thylakoid membranes.

Lipid-protein interactions in thylakoid membranes, and in the subthylakoid membrane fractions containing either photosystem 1 or photosystem 2, have been studied by using spin-labeled analogues of the thylakoid membrane lipid components, monogalactosyldiacylglycerol, phosphatidylglycerol, and phosphatidylcholine. The electron spin resonance spectra of the spin-labeled lipids all consist of two components, one corresponding to the fluid lipid environment in the membranes and the other to the motionally restricted membrane lipids interacting directly with the integral membrane proteins. Spectral subtraction has been used to quantitate the fraction of the membrane lipids in contact with the membrane proteins and to determine the selectivity between the different lipid classes for the lipid-protein interaction. The fractions of motionally restricted lipid in the thylakoid membrane are 0.36, 0.39, and 0.53, for the spin-labeled monogalactosyldiacylglycerol, phosphatidylcholine, the phosphatidylglycerol, respectively. Spin-labeled monogalactosyldiacylglycerol exhibits very little preferential interaction over phosphatidylchline, which suggests that part of the role of monogalactosyldiacylglycerol in thylakoid membranes is structural, as is the case for phosphatidylcholine in mammalian membranes. Spin-labeled phosphatidylglycerol shows a preferential interaction over the corresponding monogalactosyldiacylglycerol and phosphatidylcholine analogues, in contrast to the common behavior of this lipid in mammalian systems. This pattern of lipid selectivity is preserved in both the photosystem 1 and photosystem 2 enriched subthylakoid membrane fractions.

Chemical Phenomena↗
PubMed1989

Rotational motion of yeast cytochrome oxidase in phosphatidylcholine complexes studied by saturation-transfer electron spin resonance.

Cytochrome oxidase from yeast has been covalently labeled with a nitroxide derivative of maleimide and reconstituted in lipid-substituted complexes with dimyristoyl-, dioleoyl-, or dielaidoyl-phosphatidylcholine. The rotational mobility of the enzyme in the complexes has been studied as a function of temperature and time, and of lipid/protein ratio, using saturation-transfer electron spin resonance spectroscopy. For complexes with dimyristoylphosphatidylcholine, the rotational mobility of the protein decreases abruptly below the gel-to-fluid-phase transition. This change is accompanied by a lateral segregation of the protein, as seen by freeze-fracture electron microscopy, and by an increase in the activation energy for the enzymatic activity. A time-dependent decrease in the rotational motion of the protein is observed on incubating at temperatures in the fluid phase of the lipid. This corresponds with a time-dependent loss of enzyme activity observed on incubation at temperatures in the fluid phase, but not at temperatures in the gel phase, over a period of 3 h. The rotational mobility decreases with increasing protein concentration in the complexes, both in the fluid and in the gel phases. The dependence of the protein mobility on lipid/protein ratio can be interpreted quantitatively in terms of the effect of increased random protein-protein contacts in the fluid phase. The maximum limiting rotational correlation time for the protein diffusion at high lipid/protein ratios in the fluid phase is tau R[[ approximately equal to 25 microseconds, suggesting that the protein is present as either a monomer or more probably a dimer in the reconstituted membrane.

Dimyristoylphosphatidylcholine↗
PubMed1989

Polymorphic phase behavior of lysopalmitoylphosphatidylcholine in poly(ethylene glycol)-water mixtures.

The polymorphic phase behavior of 1-palmitoyl-2-lyso-sn-glycero-3-phosphocholine dispersions in excess water has been studied as a function of temperature and poly(ethylene glycol) (PEG) concentration, using proton dipolar-decoupled 31P NMR spectroscopy and turbidity measurements. The phase behavior was found to depend on both lipid concentration and PEG concentration, and most of the NMR experiments were conducted at a lipid concentration of 15 mg/mL. At low PEG concentrations (0-12 wt %), a thermotropic transition occurs at 3-5 degrees C with increasing temperature, from an interdigitated lamellar gel (L beta i) phase to a normal micellar phase. At intermediate PEG concentrations (12-20 wt %), thermotropic transitions take place with increasing temperature, first from the lamellar gel phase to a fluid cubic (Q alpha) phase and then at higher temperatures from the cubic phase to the micellar phase. At intermediate PEG concentrations above the former range (20-30 wt %), thermotropic transitions take place with increasing temperature, first from the lamellar gel phase to the cubic phase, then from the cubic phase to a normal hexagonal (HI) phase, and finally from the hexagonal phase to the micellar phase. At high PEG concentrations (greater than 30 wt %), a thermotropic transition takes place with increasing temperature from the lamellar gel phase directly to the fluid hexagonal phase. At these high PEG concentrations, the micellar phase is not attained within the accessible temperature range (less than or equal to 90 degrees C).(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetics↗
PubMed1989

Effect of acyl chain composition on salt-induced lamellar to inverted hexagonal phase transitions in cardiolipin.

Salt-induced fluid lamellar (L alpha) to inverted hexagonal (HII) phase transitions have been studied in diphosphatidylglycerols (cardiolipins) with different acyl chain compositions, using 31P nuclear magnetic resonance (NMR) spectroscopy. Cardiolipins with four myristoyl chains, tetramyristoyl cardiolipin (TMCL), and with four oleoyl chains, tetraoleoyl cardiolipin (TOCL), were synthesized chemically. TMCL was found to undergo a thermotropic lamellar gel to lamellar liquid-crystalline phase transition at 33-35 degrees C. This lipid exhibited an axially symmetric 31P-NMR spectrum corresponding to a lamellar phase at all NaCl concentrations between 0 and 6 M. In the case of TOCL, formation of an HII phase was induced by salt concentrations of 3.5 M NaCl or greater. These observations, taken together with earlier findings that bovine heart cardiolipin aqueous dispersions adopt an HII phase at salt concentrations of 1.5 M NaCl or greater, indicate that increasing unsaturation and length of the acyl chains favour formation of the HII phase in diphosphatidylglycerols.

Calorimetry, Differential Scanning↗
PubMed1989

A novel spin-label for study of membrane protein rotational diffusion using saturation transfer electron spin resonance. Application to selectively labelled class I and class II-SH groups of the shark rectal gland Na+/K+-ATPase.

Na+/K+-ATPase in membranous preparations from the rectal gland of Squalus acanthias has been spin-labelled either on Class I -SH groups, which maintain overall ATPase activity, or on Class II -SH groups, for which only phosphorylation activity is preserved. Labelling of the Class I groups requires solubilization of the membranes and subsequent reconstitution by precipitation with Mn2+ in order to remove contaminating peripheral proteins, which are also labelled. Control experiments with preparations in which the Class II groups are labelled demonstrate that the mobility and aggregation state of the enzyme in the reconstituted membranes are similar to those in the native membrane. Both the conventional maleimide nitroxide derivative and a new benzoylvinyl nitroxide derivative have been used for the labelling. The segmental mobility of the labels and the overall rotational diffusion of the labelled protein have been investigated using saturation transfer ESR spectroscopy. The benzoylvinyl spin-label derivative offers particular advantages for the study of the protein rotational mobility in that the segmental mobility is considerably reduced relative to that observed with the maleimide derivative. This is especially the case for the Class I groups, where the maleimide label exhibits pronounced segmental mobility. Comparison of the results from the two labels indicates that the integral of the saturation-transfer spectrum is much more sensitive to segmental motion than are the diagnostic line-height ratios. This fact allows a better level of discrimination between the two types of motion. The results from the benzoylvinyl nitroxide-labelled Class I groups suggest that the Na+/K+-ATPase is probably present as an (alpha beta)2-diprotomer (or higher oligomer) in the native membrane.

Animals↗
PubMed1989

Rotational diffusion of mitochondrial ADP/ATP carrier studied by saturation-transfer electron spin resonance.

The rotational mobility of the mitochondrial ADP/ATP carrier has been studied solubilized in Triton micelles, reincorporated in phospholipid liposomes, and in mitochondria. Spin-labeled analogues of the noncovalent inhibitors carboxyatractyloside and atractyloside were found to be strongly immobilized when bound to the carrier [Munding, A., Beyer, K., & Klingenberg, M. (1983) Biochemistry 22, 1941-1947], such that saturation-transfer electron spin resonance spectroscopy could be used to study the rotational motion of the protein. Spin-labeled maleimide covalently bound to the carrier was found to have independent segmental motion and hence to be unsuitable for studies of protein rotation. The ESR spectra of the carboxyatractyloside and atractyloside spin labels were found to contain a second component from label in the lipid or detergent, necessitating the use of the saturation-transfer ESR integral method [Horváth, L. I., & Marsh, D. (1983) J. Magn. Reson. 54, 363-373] to analyze the rotational motion of the label component bound to the protein. Effective rotational correlation times obtained from integration of the high-field region of the spectrum were lower than those obtained from the total spectral integral, indicating strongly anisotropic rotational diffusion of the carrier in the vesicular and membrane systems, with the spin-label z axis oriented preferentially perpendicular to the rotation axis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Chain configuration and flexibility gradient in phospholipid membranes. Comparison between spin-label electron spin resonance and deuteron nuclear magnetic resonance, and identification of new conformations.

The electron spin resonance spectra of 1-myristoyl-2-[n-(4,4-dimethyloxazolidine-N-oxyl)myristoyl]-sn- glycero-3-phosphocholine spin-label positional isomers (n = 6, 10, and 13) have been studied in oriented, fully hydrated bilayers of dimyristoylphosphatidylcholine, as a function of temperature and magnetic field orientation. The spectra have been simulated using a line-shape model which incorporates chain rotational isomerism, as well as restricted anisotropic motion of the lipid molecules as a whole, and which is valid in all motional regimes of conventional spin-label electron spin resonance (ESR) spectroscopy. At least one component of the lipid motion is found to lie in the slow-motion regime for all label positions, even in the fluid liquid crystalline phase, well above the phase transition. In the gel phase, the chain isomerism lies in the slow-motional regime, and the overall motions are at the rigid-limit. In the fluid phase, the chain isomerism is in the fast-motional regime, and the chain axis motions are in the slow regime. This indicates that the commonly used motional-narrowing theory is not appropriate for the interpretation of spin-label spectra in biological membranes. The simulation parameters yield a consistent description for the chain order and dynamics for all label positions. The correlation times and order parameters for the overall motion are the same at all positions down the chain, whereas the chain conformation and trans-gauche isomerism rate display a characteristic flexibility gradient, with increasing motion towards the terminal methyl end of the chain. Significantly, it is found that all six distinct tetrahedral orientations of the hyperfine tensor at the labeled segment are required for a consistent description of the chain isomerism. For the C-6 segment only the 0 degree (trans) and two 60 degrees (gauche) orientations are significantly populated, for the C-10 position two further 60 degrees orientations are populated, and for the C-13 position all orientations have non-vanishing populations. Detailed comparisons have been made with the results of 2H nuclear magnetic resonance (NMR) measurements on dimyristoylphosphatidylcholine labeled at the same position in the sn-2 chain, and using an identical motional model. The parameters of overall reorientation, both order parameter and correlation times, have very similar values as determined by ESR and NMR. The major difference between the results from the two methods lies in the conformational populations at the labeled chain segment and the trans-gauche isomerization rate in the gel phase. The conformational order is much lower for the spin-labeled chain segments than for the corresponding deuterium-labeled segments, and the isomer interconversion rates in the gel phase(although displaying a mobility gradient in both cases) are found to be much slower in the former case. In addition the spin-label measurements provide information on the macro order (chain tilt), which is only available from oriented samples. These results are consistent between the different spin label positions and are in agreement with the findings from x-ray diffraction.

Dimyristoylphosphatidylcholine↗
PubMed1989

Water adsorption isotherms and hydration forces for lysolipids and diacyl phospholipids.

The repulsive forces in a wide range of diacyl and monoacyl phospholipid systems have been obtained from the adsorption isotherms for water. From the exponential dependence of the repulsive pressure on the water content, information has been deduced regarding the hydration force. For diacyl phosphatidylcholines the strength of the hydration force and its characteristic decay length are in good agreement with values previously obtained by x-ray diffraction methods. For natural and synthetic diacyl phosphatidylcholines in the fluid lamellar phase, the hydration force extrapolated to zero layer separation (Po) is in the range 4-5.10(8) N.m-2 and the decay length is approximately 0.3 nm. The results for dimyristoyl, dipalmitoyl, and distearoyl phosphatidylcholines in the gel phase are very similar with Po approximately 2.5.10(8) N.m-2 and decay length of approximately 0.2 nm. Egg monomethyl phosphatidylethanolamine is less strongly hydrated: Po = 2.3.10(9) N.m-2, with a decay length of 0.3 nm. Egg phosphatidylethanolamine and bovine phosphatidylserine hydrate even more weakly with Po approximately 1.3.10(8) N.m-2 and decay length of approximately 0.15 nm. Mixtures with cholesterol or phosphatidylcholine increase both Po and the decay length for phosphatidylethanolamine to values closer to those for phosphatidylcholine. The repulsive forces deduced for egg lysophosphatidylcholine at 40 degrees C display a biphasic water dependence, with the low water phase being similar to lamellar egg phosphatidylcholine, and the phase at higher water content having a smaller value of Po = 2.10(8) N.m-2 but a longer decay length of approximately 0.45 nm, corresponding to a nonlamellar configuration. Bovine lysophosphatidylserine similarly yields values of PO = 1.2.108 N.m-2 and an effective decay length of 0.64 nm. The hydration behavior of the various diacyl phospholipids has been interpreted in terms of the mean-field molecular force theory of lipid hydration, and values deduced for the surface hydration potential of the various lipids. This analysis extends previous results on hydration forces, particularly to lysolipids and nonlamellar phases.

Cholesterol↗
PubMed1988

Influence of lipid headgroup on the specificity and exchange dynamics in lipid-protein interactions. A spin-label study of myelin proteolipid apoprotein-phospholipid complexes.

The pH and salt dependences of the interaction of phosphatidic acid, phosphatidylserine, and stearic acid with myelin proteolipid apoprotein (PLP) in dimyristoylphosphatidylcholine (DMPC) recombinants have been studied by electron spin resonance spectroscopy, using spin-labeled lipids. The two-component spin-label spectra have been analyzed both by spectral subtraction and by simulation using the exchange-coupled Bloch equations to give the fraction of lipids motionally restricted by the protein and the rate of lipid exchange between the fluid and motionally restricted lipid populations. For stearic acid, phosphatidic acid, and phosphatidylserine, the fraction of motionally restricted spin-label increases with increasing pH, with pKa's of 7.7, 7.6, and ca. 9.4, respectively. The corresponding pKa's for the bulk lipid regions of the bilayer are estimated, from changes in the ESR spectra, to be 6.7, 7.4, and 11, respectively. In the dissociated state at pH 9.0, the fraction of motionally restricted component decreases with increasing salt concentration, reaching an approximately constant value at [NaCl] = 0.5-1.0 M for all three negatively charged lipids. The net decreases for stearic acid and phosphatidic acid are considerably smaller (by ca. 30%) than those obtained on protonating the two lipids, whereas for phosphatidylserine the fraction of motionally restricted lipid in high salt is reduced to that corresponding to phosphatidylcholine. For a fixed lipid/protein ratio, the on-rate for exchange at the lipid-protein interface is independent of the degree of selectivity and has a shallow temperature dependence, as expected for a diffusion-controlled process.(ABSTRACT TRUNCATED AT 250 WORDS)

Algorithms↗
PubMed1988

Spin label studies on the selectivity of lipid-protein interaction of cardiolipin analogues with the Na+/K+-ATPase.

The selectivity of lipid-protein interaction for various spin-labelled cardiolipin analogues in Na+/K+-ATPase membranes from Squalus acanthias has been investigated by ESR spectroscopy. Cardiolipin derivatives with different numbers of acyl chains, or in which the headgroup charge has been removed by methylation of the phosphate groups, all show a pronounced selectivity relative to phosphatidylcholine. Maximally three times more of the cardiolipin analogue is associated with the protein, than is phosphatidylcholine. The selectivity pattern in the absence of salt is in the order: cardiolipin approximately monolysocardiolipin greater than or equal to acylcardiolipin greater than dimethylcardiolipin much greater than phosphatidylcholine, where acylcardiolipin has the spin label chain attached to the centre-OH group of the headgroup. The degree of association of the negatively charged cardiolipins with the protein is reduced by salt, corresponding to the lower selectivity for dimethylcardiolipin. It is concluded that the selectivity of the Na+/K+-ATPase for cardiolipin is not solely of electrostatic origin, nor is it likely to originate in the larger number of fatty acid chains relative to diacyl phospholipids.

Animals↗
PubMed1988

Novel spin-labels for the study of lipid-protein interactions. Application to (Na+, K+)-ATPase membranes.

The interactions of a series of spin-labeled fatty acids, in which the nitroxide ring is incorporated in different ways as an integral part of the hydrocarbon chain, with the (Na+,K+)-ATPase in membranes from Squalus acanthias, have been studied by electron spin resonance spectroscopy. The fatty acids are 2,4-, 2,5-, and 3,2-substituents of 2,2,5,5-tetramethylpyrrolidine-N-oxyl and belong to the class of minimal perturbation nitroxide probes. For all five fatty acid labels, a motionally restricted lipid component was observed in the ESR spectra of (Na+,K+)-ATPase membranes, in addition to the fluid component, which was found in the spectra of the extracted membrane lipids. The pH dependence of the motionally restricted spin-label population indicated a sensitivity in the selectivity of the lipid-protein interaction to the protonation state of the fatty acid. These results agree with those found previously for the conventional oxazolidine (doxyl) fatty acid and phospholipid spin-label derivatives [Esmann, M., Watts, A., & Marsh, D. (1985) Biochemistry 24, 1386-1393] and indicate that the motion of the lipid chains is significantly hindered by interaction with the protein, irrespective of the nature of the spin-label group.

Animals↗
PubMed1988

Ganglioside-protein interactions: spin-label electron spin resonance studies with (Na+,K+)-ATPase membranes.

Lipid-protein interactions in (Na+,K+)-ATPase-rich membranes from Squalus acanthias have been studied using spin-labeled derivatives of the mono- and disialogangliosides GM1, GM2, GM3, and GD1b, in conjunction with electron spin resonance (ESR) spectroscopy. Ganglioside-protein interactions are revealed by the presence of a second component in the ESR spectra of the membranes in addition to a component that corresponds closely to the ESR spectra obtained from dispersions of the extracted membrane lipids. This second component corresponds to spin-labeled gangliosides whose chain motion is significantly restricted relative to that of the fluid lipids in the membrane or the lipid extract. A small selectively for the motionally restricted component associated with the protein is found in the order GD1b greater than GM1 approximately equal to GM2 approximately equal to GM3. Comparison with previous results from spin-labeled phospholipids in the same system [Esmann, M., Watts, A., & Marsh, D. (1985) Biochemistry 24, 1386-1393] shows that the spin-labeled monosialogangliosides GM1, GM2, and GM3 display little selectivity in the lipid-protein interaction relative to spin-labeled phosphatidylcholine. The spectral characteristics of both the fluid and motionally restricted spin-labeled components differ very significantly, however, between the gangliosides and the phospholipids. The outer hyperfine splitting of the motionally restricted component is smaller for the gangliosides than for the phospholipids, indicating a smaller degree of motional restriction on interaction of the ganglioside lipid chains with the protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Exchange rates at the lipid-protein interface of myelin proteolipid protein studied by spin-label electron spin resonance.

The electron spin resonance (ESR) spectra from spin-labeled phospholipids in recombinants of myelin proteolipid apoprotein with dimyristoylphosphatidylcholine have been simulated with the exchanged-coupled Bloch equations to obtain values for both the fraction of motionally restricted lipids and the exchange rate between the fluid and motionally restricted lipid populations. The rate of exchange between the two spin-labeled lipid components is found to lie in the slow exchange regime of nitroxide ESR spectroscopy. The values obtained for the fraction of motionally restricted component in the exchanged-coupled spectra are found to be in good agreement with those obtained previously by spectral subtraction for the same system [Brophy, P. J., Horváth, L. I., & Marsh, D. (1984) Biochemistry 23, 860-865]. The rate of lipid exchange off the protein is independent of lipid/protein ratio for a given spin-labeled phospholipid, as expected, and decreases with increasing selectivity of the various phospholipids for the protein. At 30 degrees C and for ionic strength 0.1 and pH 7.4, the off-rate constants are 4.6 X 10(6) s-1 for phosphatidic acid, 1.1 X 10(7) s-1 for phosphatidylserine, 1.6 X 10(7) s-1 for phosphatidylcholine, and 2.2 X 10(7) s-1 for phosphatidylethanolamine. These values are in the inverse ratio of the relative association constants of the various lipids for the protein (Brophy et al., 1984) and are appreciably slower than the rate of lipid lateral diffusion in dimyristoylphosphatidylcholine bilayers.(ABSTRACT TRUNCATED AT 250 WORDS)

Dimyristoylphosphatidylcholine↗
PubMed1988

Promoting occupational therapy in the schools.

In response to our perceptions of the need to clarify the nature of school-based occupational therapy services at the local, state, and federal levels, we have written this article to address three areas. First, we suggest that the terminology used by school-based occupational therapists to describe their work be explained in terms that will allow parents and educators to better understand the roles and functions of the occupational therapist in the school. Second, to clarify and thereby increase the marketability of school-based occupational therapy, we propose a conceptual framework upon which the provision of occupational therapy as a service related to education can be based. Third, to clarify the unique role of occupational therapy in the schools, we define the roles and functions of school-based occupational therapy in such a way as to differentiate it from other related school-based services. Finally, we identify strategies to further secure the position of occupational therapy in the schools.

Persons with Disabilities↗